Plasmodium falciparum and the urgency of therapeutic management, quick and reliable diagnosis is required for early detection. Blood smear microscopy remains the gold standard for malaria diagnosis. Molecular diagnosis techniques are the most sensitive and specific in cases of low parasitaemia and in the detection of mixed infections. The purpose of this study was to evaluate a new commercial test involving the molecular diagnostic technique to detect the five human Plasmodium species. The Vitassay qPCR Malaria 5 test is based on the multiplex real-time PCR of a conserved target region of the 18S rRNA gene for the five human Plasmodium species. A total of 190 samples collected from imported cases of malaria were diagnosed using this test and compared against a homemade reference real-time PCR. The sensitivities of the Vitassay qPCR Malaria 5 test for all Plasmodium species ranged from 93.8% to 100% and specificity ranged from 97.7% to 100%. Based on these criteria, this test is recommended for the diagnosis of the human Plasmodium species.Malaria is the most common human parasitic disease in the world with the highest morbidity and mortality. Due to the severity of malaria caused by  Malaria is one of the most severe public health problems worldwide, causing an estimated 241 million clinical cases and leading to 627,000 deaths in 2020 [Plasmodium falciparum (88.5%). It is imperative that the Malaria CNR has the most efficient diagnostic tools. Effective diagnostic techniques could enable the early diagnosis of malaria, and prevent infection from progressing to severe malaria or death. Diagnosis should be performed within four hours of the sample’s arrival in metropolitan France [P. falciparum [P. falciparum histidine-rich protein 2 (PfHRP2)-based RDTs, due to parasites lacking the pfhrp2 gene [P. falciparum, P. vivax, P. malariae, and P. ovale). It is essential to select the most efficient kit depending on test performance (sensitivity and specificity) and operating constraints (storage conditions and ease of use).France is the non-endemic European country most affected by imported cases of malaria. The French National Reference Centre for Malaria  is charged with the epidemiologic surveillance of imported malaria cases in France . Some 21n France . As recolciparum ,5. Moreorp2 gene . Over thrp2 gene . A homemrp2 gene . CurrentThe aim of this study was to evaluate the sensitivity and specificity of the Vitassay qPCR Malaria 5 test, based on the real-time PCR of the 18S rRNA plasmodial gene. A thin blood smear, the gold-standard method, and a homemade real-time PCR were used as references.The samples were taken from malaria cases imported from endemic countries in patients hospitalised in French civilian or military hospitals and sent to the Malaria CNR .P. falciparum, and mixed infections, as well as 100 complementary isolates collected between 2021 and 2022 without selection.Ninety samples isolated between 2019 and 2020 were assessed in this study  and retrPlasmodium infection samples were identified from thin blood smears stained with eosin and methylene blue using a RAL® kit  by well-trained and experienced technicians. The parasitaemia percentage was estimated by counting the number of infected cells as a percentage of the red blood cells.Plasmodium species (with the exception of P. knowlewsi), as previously described [® DNA Blood Mini kit, according to the manufacturer’s recommendations . Each parasite species was detected by an individual real-time PCR targeting a specific gene for each of the four human Plasmodium species using the Light Cycler® TaqMan® Master Mix . For each PCR run, two negative controls (ultra-pure water and human DNA) and one positive control (specific DNA from each species) were used.The reference molecular diagnosis was performed by real-time PCR using a Light Cycler 2.0  to identify four human escribed . The priP. ovale wallikeri from P. ovale curtisi using Plasmo1_F (5′ GTTAAGGGAGTGAAGACGATCAGA 3′) and Plasmo2_R (5′ AACCCAAAGACTTTGATTTCTCATAA 3′) primers, as previously described [Homemade real-time PCR tests, targeting the 18S rRNA gene, were performed to differentiate Plasmodium species. The first low-profile strip contains the multiplex reaction mix for the detection of P. malariae, P. knowlesi, and/or P. ovale, while the second contains the mix for the detection of P. falciparum and/or P. vivax. Each plasmodial species was simultaneously detected by distinct fluorescence channels: P. malariae and P. falciparum DNA targets in the FAM channel, P. knowlesi and P. vivax DNA targets in the ROX channel, and P. ovale DNA targets in the Cy5 channel, and the EC DNA target is amplified and detected in the HEX channel. For each PCR run, the EC, a negative control (human DNA), and a positive control (specific DNA from each species) were included. The EC allows the detection of a problem of extraction of a poor-quality and/or a shoddy reaction due to inhibitor presence.The Vitassay qPCR Malaria 5 test is a ready-to-use multiplex PCR that can be used with the CFX96™ Real-Time PCR Detection System , according to the manufacturer’s recommendations . DNA was extracted from 200 µL of whole blood using an extraction control (EC). This test contains, in each well, all the necessary real-time PCR reagents to amplify a conserved target region of the 18S rRNA gene for the five human Plasmodium species infection against the reference homemade real-time PCR results. Diagnostic performances such as sensitivity and specificity were evaluated using the following formula :The aim of analysing the results was to compare the ability of the Vitassay qPCR Malaria 5 kit to detect each t test was used appropriately, p-values were evaluated, and statistical significance was set at p-values < 0.05.For statistical analysis, the Fisher Student’s All the samples were detected by thin blood smears and confirmed by real-time PCR.P. falciparum (parasitaemia between <0.001% and 33%) by homemade real-time PCR, 5 as P. vivax (parasitaemia between <0.001% and 0.6%), 11 as P. malariae (parasitemia between <0.001% and 0.2%), 16 as P. ovale (parasitemia between <0.001% and 0.15%), 8 as mixed infections , and 3 as negative samples  and the homemade reference real-time PCR, only three discrepant diagnoses were observed. One case was due to fections .Plasmodium species . Samples with low parasitaemia  were correctly detected. Overall, mixed infections with P. falciparum and non-falciparum species were distinctly diagnosed. The two species of P. ovale, wallikeri, and curtisi were successfully identified.The 187 other diagnoses showed no significant difference in terms of the specific detection of the four human P. falciparum, 100% for P. vivax, 100% for P. ovale, and 93.8% for P. malariae, while the respective specificities were 97.7% for P. falciparum, 100% for P. vivax, 99.4% for P. ovale, and 100% for P. malariae   and the specific target detected. However, the Ct value results between the two real-time PCR techniques were concordant with Pearson’s correlation coefficient (r) of 0.984 and a × 10−91) .The Ct values of EC included in each reaction were constant, which made it possible to validate the performance and reproducibility of the Vitassay qPCR Malaria 5 test.p-value = 1).The diagnostic performance between the Vitassay qPCR Malaria 5 test and the reference homemade real-time PCR was compared in 190 clinical samples using a Fisher’s exact test. There was no significant difference between the two assays  of the Vitassay qPCR Malaria 5 test for P. falciparum by multiple dilutions of an isolate. This LOD was estimated at a parasitaemia value equivalent to 0.000001%, while the LOD of the microscopy was estimated at 0.001% . The analytical specificity for malaria pathogens was tested within a panel of different microorganisms, and no cross-reactivity was observed between any of these species. In addition, the test was highly specific for all the individual Plasmodium spp.The Vitassay qPCR Malaria 5 test makes it possible to detect and differentiate To confirm the appropriate performance of the molecular diagnostic technique, an EC was included in each reaction. The emission of the EC signal makes it possible to confirm the validity of the extraction procedure and the absence of any nonspecific amplification and/or contamination.Moreover, the freeze-dried reagents and the test can be shipped and stored at temperatures between 2 °C and 40 °C until the expiration date indicated on the label, a feature which is essential under restrictive conditions.For early management and treatment, the reliable diagnosis of malaria remains a vital means of specifically detecting the presence of malarial parasites in cases of fever in endemic malarial regions. The time required for a complete molecular diagnosis according to the two techniques is less than three hours. Moreover, the Vitassay qPCR Malaria 5 test is performed with a lower risk of contamination in comparison with the homemade reference real-time PCR.This kit was efficient in detecting malaria parasites with sensitivities ranging from 93.75% to 100% and specificities ranging from 97.7% to 100%. In malaria samples where parasitaemia was very low (<0.001%), the molecular diagnosis remained reliable.P. falciparum is known to lead to severe malaria. By contrast, P. ovale, P. vivax, or P. malariae cause less severe infections. Cases of P. vivax severe infections have been reported at low prevalence in the last decade. Adequate diagnosis for appropriate treatment (artemisinin-based combination therapy followed by primaquine) leads majorly to prevent relapse in cases of co-infection of P. falciparum with P. vivax or P. ovale [P. falciparum [P. falciparum mono-infection [There was no significant difference in the detection of mixed malaria infections. The major advantage of using this assay would be to detect mixed infections, as is the case in French Guiana and the Republic of Djibouti ,10,11. MP. ovale ,15. UncoP. ovale . The rislciparum . In concnfection .P. ovale parasites, there was no difference in diagnosis between the two species, P. ovale curtisi and P. ovale wallikeri. The majority of imported malaria cases in metropolitan France diagnosed by the Malaria CNR come from Africa, where P. falciparum is predominant, but also from specific African countries  where the transmission of P. ovale has been reported [P. falciparum, 29.0% P. vivax, 7.2% P. ovale, and 1.8% P. malariae [P. vivax is predominant [In the context of reported ,20,21. Bmalariae . MoreoveP. knowlewsi.The limitations of this study include the low number of samples. Although 190 malaria samples were tested, given the African origin of these samples, none were identified as Diagnostic tools for malaria have expanded beyond blood smear microscopy, previously the gold-standard technique. Molecular and innovative techniques have emerged, including RDTs and methods derived from PCR such as nested PCR, real-time PCR, and loop-mediated isothermal amplification (LAMP) . These mThe Vitassay qPCR Malaria 5 test has been shown to be a reliable tool for the diagnosis of malaria in non-endemic countries, detecting low parasitaemia and mixed infections with a sensitivity similar to the homemade reference real-time PCR methods, but is more practical to use due to its multiplex nature.


Malaria is the leading vector-borne parasitic disease that is causing high morbidity and mortality worldwide. So far huge efforts to control and eliminate malaria are hindered by the occurrence of asymptomatic carriers that are a potential source of infection. Yet, there is a scarcity of data nationally and in the current study area as well. Therefore, this study was aimed to assess the prevalence of asymptomatic malaria in Northeast Ethiopia.A community-based cross-sectional study was conducted in 2019 involving a total of 270 study participants recruited via purposive non-probability sampling technique. A structured questionnaire was used to collect data on sociodemographic characteristics, individual and household factors related to asymptomatic malaria. Data were entered in Epi Data 3.1 version and analyzed by using SPSS version 20, and p< 0.05 was considered statistically significant.The overall prevalence of asymptomatic malaria was 7.0%, with 3.0%, 5.2%, and 12.0%, respectively by Rapid diagnostic tests (RDT), Microscopy and Polymerase chain reaction (PCR). The majority of infections (73.7%) were identified from index households. Previous malaria history , living with index cases  and family size > 6 members  were significant predictors of asymptomatic malaria.Reactive case detection had identified considerably higher asymptomatic malaria cases in the community. Therefore, active case investigation should be established in the community by tracking the symptomatic cases at the health facilities. Plasmodium, and transmitted through the bite of infected female Anopheles mosquitoes. Malaria is a serious public health problem in many parts of the world, exacting an unacceptable toll on the health and economic welfare of the world's poorest communities. Based on the WHO report, there were an estimated 228 million malaria episodes, and 405 000 malaria deaths in 2020, of which approximately 90% of the burden resided in Africa. Approximately 90 % of malaria deaths occurred in children under 5 years of age and pregnant women  are the most widely used diagnostic methods for clinical malaria and routine surveillance . HoweverMalaria control activities have substantially reduced malaria incidence over the last decade through different strategic interventions, including environmental management, indoor residual spraying, long-lasting insecticide-treated nets, and early diagnosis and prompt treatment . EthiopiThe presence of high prevalence asymptomatic carriage poses a threat to malaria elimination and control programs . Since aAnopheles mosquitoes increase in number by harboring the gametocyte stage to continue the cycle  of Ethiopia, the district had a total of 221,958 inhabitants  . The disStudy participants: The study participants for this particular research work were household contacts and neighbors of laboratory-confirmed malaria cases that lived at least for a year in the area. However, children below 6 months, individuals who had taken anti-malaria drugs within 4 weeks prior to data collection, and members of households and neighbors of laboratory-confirmed malaria cases with clinical signs and symptoms of malaria  were excluded.Sample size and sampling technique: A sample size of 270 was calculated using single population proportion statistical formula; by the following assumptions: 95% level of confidence, 3.5 % margin of error and P (proportion) of 95 % taken from a previous study , and household level factors  related to Plasmodium spp infection such as residence, family size via a face-to-face interview by the principal investigator.Blood sample collection, handling, and transportation: Finger prick blood specimen was collected for dried blood spot (DBS), RDT, and blood film. Three to four drops of blood were immediately spotted on Whatman 3MM filter paper for molecular analysis. The DBS was packaged individually into a double zip-lock plastic bag with desiccant and subsequently transported to Woldia General Hospital Laboratory for storage at - 20 °C. Later, it was transferred to Amhara Public Health Institute for PCR analysis.RDT  Ag Combo Test): After the pouch is open for immediate use, the collected capillary sample was blotted onto the sample pad of the test kit. Approximately, two drops of buffer were added onto the buffer pad and the result was read at the end of 20 minutes.Blood films: Both thin and thick blood films were prepared for the microscopic investigation of Plasmodium spp. The thin blood smear was fixed by carefully dropping methanol using a Pasteur pipette immediately after drying. The methanol-fixed thin smears were allowed to dry completely in the air (approximately 2 min) by placing the slides on a flat surface. The prepared blood smears were transported to the nearby health center in a slide box for Giemsa staining DNA Extraction: Briefly, three 3-mm punches of the DBS were punched out and placed into a 1.5-mL Eppendorf tube. The punches inside the Eppendorf tube were treated with ATL buffer, proteinase K, and AL buffer with brief vortex and incubation with respective addition. The mixtures were transferred into the QIAamp Mini spin column and with the subsequent addition of wash buffer AW1 and AW2. The DNA was eluted in 100 µL of elution buffer, aliquoted, and stored at -20°C until use.DNA amplification: Amplification of the Genus and P. falciparum multiplex was performed in a 20-ml reaction tube containing 10.0 µl 2X ABI TaqMan buffer, 0.5 µl each forward and reverse primer for the Genus, 0.5 µl forward and 0.25 µl reverse primer P. falciparum and 5 µl of DNA template. The reactions were performed under the following cycling parameters: initial hot-start at 95° C for 15 minutes, followed by 45 cycles of denaturation at 95 °C for 20 seconds, and 45 cycles annealing at 60 °C for 40 seconds. The correct fluorescence channel was selected for each fluorescently labeled primer set and the cycle threshold values were recorded at the end of the annealing step. Any sample with a cycle threshold value of 40 or below was considered positive. Both singleplex and multiplex assays were performed for all the samples  version 20  for analysis. Bivariable and multivariable logistic regression was performed to assess the association between the dependent variable and independent variables. Variables with a P-value of < 0.25 in the bivariable analysis were included in the multivariable logistic regression analysis. The odds ratio was calculated with a confidence interval of 95%, and a P-value < 0.05 was considered statistically significant.Socio-demographic characteristics: A total of 270 study participants residing in 60 households were enrolled in the study (72 from 20 index houses and 198 from 40 neighboring houses). The majority (61%) of the participants were female. The mean age of the participants was 21.02 (+18.6 SD) years. About 38.9%, 32.6%, and 17.0% of the participants were from Addis Kign Aradom, and Jarota  and P. vivax 42.1% (8/19) were identified in this study. All the 270 blood samples collected were tested for malaria via the rapid diagnostic test (RDT) and microscopy, yielding positivity rates of 3.0% (8/270), and 5.2% (14/270), respectively. However, only 92 samples were processed by the PCR method and it gave a prevalence of malaria at 12.0% (11/92). PCR detected asymptomatic malaria about 2.7-fold and 2.3-fold higher than RDT and microscopy, respectively  and Aradom (8.0%)  and 2.5% (5/198), respectively. The highest prevalence of asymptomatic malaria was recorded at two m (8.0%) .Plasmodium InfectionIndividual and Household Factors Associated with Bivariable and multivariable analyses: The bivariable analysis of individual and household factors showed a significant association with asymptomatic malaria at p-value <0.05. Consequently, age group > 15 years (COR: 3.1 [CI: 1.09–8.88]) and previous history of malaria (COR: 6.727 [1.912–23.668]) showed significant association with Plasmodium infection. Moreover, participants living in Addis Kign subdistrict (COR: 2.9 [CI: 1.11–7.66]), within index houses (COR: 5.5 [CI: 2.056–14.486]), and family size > 6 members (COR: 5.3 [CI: 5.291 (1.504–18.610]) were at higher odds of Plasmodium infection  with p ≤ 0.2 in the bivariable analysis were selected multivariate analysis to control confounder variables. After adjusting for confounders, only three variables  showed a statistically significant association with Plasmodium species infection (Eight predictors (age, relation with index cases, nfection .Plasmodium infection in individuals having previous malaria history was 4.0 times higher compared to those who didn't have it. Participants who lived in the index houses were at 3.9 times higher odds of getting Plasmodium infection than those who lived in neighboring houses. The odds of asymptomatic Plasmodium infection in households who had six or more members had 4.8 times higher than families with lower members (The odds of  members .Asymptomatic malaria becomes a serious challenge in malaria elimination. Individuals with asymptomatic malaria have low health-seeking behavior, and therefore, they remain potential reservoirs in sustaining malaria transmission .Plasmodium spp infection. This is in line with studies in Northwest Ethiopia 4.8%, (In the current study, RDT detected and identified 3% of the asymptomatic ia 4.8%, , Southeaia 4.8%, , Lao DPRia 4.8%, , and Haiia 4.8%, . On the ia 4.8%,  and Nortia 4.8%, . This diMicroscopy detected and identified 5.2% of asymptomatic infections. This is in line with studies done in Northwest Ethiopia 4.2% , 6.8% 2, WesternPlasmodium spp infections. This was higher than previous studies in Ethiopia; 8.12 % (PCR detected and identified 12% of asymptomatic ; 8.12 % , 18, Mya; 8.12 % , Zambia ; 8.12 % . However; 8.12 % , Haiti w; 8.12 % , Kenya; ; 8.12 % ,32, Indi; 8.12 % , and thi; 8.12 % . Therefo; 8.12 % .Plasmodium spp was P. falciparum with 57.9% and the remaining 42.1% was P. vivax. This finding is in line with the Ethiopian nationwide malaria figure reported by the Ministry of Health over which P. falciparum accounts for about 60% (P. vivax was the predominant species in Hadiya zone, Southern Ethiopia (Plasmodium species distribution as a result of environmental and ecological changes.This study also depicted that the predominant Ethiopia , and EasEthiopia . This diReactive case detection has identified a higher prevalence of asymptomatic infection in index houses with 19.4% compared with their neighboring houses with 3.5%. This finding is consistent with a study done in Jimma zone, Southwest Ethiopia . This diPlasmodium spp infection. This finding is supported by studies done in Ethiopia (P. falciparum in the circulation while relapse is due to the reactivation of hypnozoite which leads to initiation of the erythrocytic cycle in the case of P. vivax. However, these studies also underpinned living with index cases and family size > 6 members as potential risk factors. Reasonably, the infection is clustered in index cases' houses compared with neighbors' houses and this might be due to equal proximity to vector breeding sites or likely behaviors of family members with the index cases. As family size increases, there might be individuals who didn't have a chance to use bed nets, as universal coverage of mosquito nets is low in the country in general and the study area in particular.Regarding factors associated with asymptomatic malaria infection, previous malaria history increases 4.0 times the odds of having Ethiopia , 21, 26.


Rickettsia. While these may not be significant causes of mortality in malaria-endemic areas, they affect the daily life and performance of affected individuals. It is, therefore, important to have a clear picture of these other aetiologies to institute correct diagnoses at hospitals and improve patient outcomes.In sub-Saharan Africa, malaria is the common diagnosis for febrile illness and related clinical features, resulting in the under-diagnosis of other aetiologies, such as arboviruses and Rickettsia africae, and Plasmodium spp. using high-throughput real-time PCR techniques. A logistic regression was performed on the results to explore the effect of demographic and socio-economic independent variables on malaria infection.Blood samples were collected from patients with fever and other clinical features associated with febrile illness at selected hospitals in the malaria-endemic counties of Busia, Bungoma, and Kakamega, and screened for Crimean-Congo haemorrhagic fever, Sindbis, dengue and chikungunya viruses, Plasmodium falciparum and 1.5% (5/336) for Plasmodium malariae. Two patients had dual P. falciparum/P. malariae infections. The most common clinical features reported by the patients who tested positive for malaria were fever and headache. None of the patients were positive for the arboviruses of interest or R. africae. Patients living in Busia  and Bungoma counties  had higher odds of being infected with malaria, compared to those living in Kakamega County.A total of 336 blood samples collected from hospital patients between January 2018 and February 2019 were screened, of which 17.6% (59/336) were positive for R. africae cases in this study may have been due to the limited number of samples screened, low-level circulation of arboviruses during inter-epidemic periods, and/or the use of PCR alone as a detection method. Other sero-surveys confirming their circulation in the area indicate that further investigations are warranted.The reported malaria prevalence is in line with previous studies. The absence of arboviral and The online version contains supplementary material available at 10.1186/s12936-022-04287-3. Rickettsia spp., are known to cause mild non-pathognomonic febrile illness in humans [Rickettsia are not considered significant causes of mortality and morbidity, especially in malaria-endemic resource-poor communities. Thus, funds allocated for their study and surveillance are limited [At least a hundred arboviruses, and several n humans , 2, whicn humans . Howevern humans . The impn humans . Traditi limited , leading limited , 8.Rickettsia spp., particularly Rickettsia africae, have been widely reported in ticks collected from both the environment and cattle [R. africae), have been reported in African indigenous people [In the East African region, arboviruses are endemic, with the most important ones affecting humans being CCHF, Rift Valley fever, chikungunya, and dengue viruses . Diversed cattle –13; howed cattle . While fs people , 16, thes people –19. In Es people –22. Elses people , 24.Rickettsia [Western Kenya, comprising Busia, Bungoma and Kakamega counties, is situated at the border of Uganda and Kenya within the Lake Victoria basin of East Africa. Studies in this region have reported seropositivity to chikungunya , alphavickettsia . A singlckettsia . In termckettsia .R. africae in relation to malaria infection.Great strides have been made in Kenya to study arboviral diseases during outbreaks in the known hotspots, such as the northern and coastal areas. However, fewer investigative efforts have been undertaken during inter-epidemic periods in those areas where clinical cases have been detected and have the potential to support circulation and outbreaks. Therefore, this study was undertaken in western Kenya using a subset of blood samples collected from patients with fever and other clinical features associated with febrile illness to determine the occurrence of acute cases of CCHF, Sindbis, dengue, and chikungunya viruses and This study was carried out in the three neighbouring counties of Busia, Bungoma, and Kakamega Fig. a. Busia The blood samples used in this study were collected as part of the Zoonoses in Livestock in Kenya (ZooLinK) project, as described in detail elsewhere . The stuBriefly, a sampling frame of 24 hospitals in the three counties was created, from which a total of 12 (4 per County) were selected for sampling. In each County, a referral hospital, a missionary hospital, and two sub-County hospitals were included in the study. Each of the selected hospitals was visited once every 4 weeks for 20 sampling cycles from 2017 to 2019. At each hospital, two project clinical officers liaised with the hospital staff to ensure smooth recruitment of study participants. Up to 10 participants were recruited at each hospital per visit. The inclusion criterion was patients presenting with one or more clinical features, including fever, suggestive of an arboviral/malaria/bacterial infection  where nucleic acid extraction was performed on arrival and then stored at −80 °C for subsequent molecular analyses. The icipe ML-EID laboratory is located approximately 25 km (30–40 min) from the ILRI Nairobi laboratories.For the purpose of this study, 336 of 865 blood samples collected throughout the study, and associated demographic data were selected, based on the following criteria: (i) blood samples collected from patients with fever and other clinical features associated with febrile illness Fig.  between The magnetic-based High Prep Viral DNA/RNA kit , was used to isolate total nucleic acid from whole blood. Initially 200 µl of blood was added to 528 µl of a lysis master-mix consisting of VDR lysis buffer, isopropanol, and carrier RNA. After vortexing, 10 µl of proteinase K and 10 µl of MAG-S1 magnetic beads were added and mixed into solution by vortexing. The subsequent wash steps to separate protein and cellular debris from nucleic acid bound to magnetic particles were performed according to the manufacturer’s instructions. The High Capacity cDNA Reverse Transcription Kit  was used to synthesize cDNA in 10 µl reactions according to the manufacturer’s instructions, supplementing the random primers with 600 µM non-ribosomal random hexanucleotide primers previously described for maximum yield .R. africae, patients’ blood samples were screened with PCR-HRM using Rick-F1 and Rick-R2 primers that target the Rickettsia 16S rRNA region [To detect CCHF, Sindbis, dengue and chikungunya viruses in patients, a multiplex touchdown PCR and high-resolution melting analyses (PCR-HRM) were applied as an initial screening test to select positive samples for further identification, as described in detail by Villinger et al.  and AjamA region , 39.Plasmodium spp. using two sets of primers. Initially, a primer pair ncMS-F/ncMS-R was used to amplify a non-coding mitochondrial region (large subunit rRNA fragment E) of all Plasmodium spp. [cox 1 primers targeting a 540-bp region of the cytochrome oxidase 1 gene of Plasmodium spp. [Plasmodium falciparum DNA amplified and sequenced previously was included as a positive control in all the runs [Plasmodium spp., nucleotide sequences were queried against the GenBank nr database using BLAST .Patients’ blood samples were screened for malaria-causing ium spp.  using PCium spp. . All posium spp. . Plasmodthe runs   for binary responses. The independent variables included baseline socio-economic, demographic, and geographic variables such as county of residence, sex, age, occupation, floor type in house, livestock ownership, use of mosquito nets, and education level  and Plasmodium malariae (GenBank accession MT430946) infections were detected in patients presenting to selected hospitals in the counties under study. Two patients had dual P. falciparum/P. malariae infections , as determined by a positive PCR result for The most common clinical features in the patients who tested positive for malaria were fever and headache, followed by abdominal cramps, joint pain, and back pain in decreasing order. There were fewer occurrences of the other clinical features as reported by the patients.On univariable logistic regression analysis, county of residence, age, “mosquito nets” and education level were marginally associated with the occurrence of malaria, while the other independent variables were not Table . Therefop < 0.001) and Bungoma  counties, compared to Kakamega County  , in KisuOverall, the counties of Kisumu, Siaya, Migori, Homa Bay, Kakamega, Busia, Bungoma, and Vihiga are classified as having lake endemic malaria transmission . HoweverP. falciparum as reported by other studies with less occurrence of P. malariae and Plasmodium ovale [The most frequent species in this study was um ovale , 47, 48.The other variables in this study were not significantly associated with malaria infection. Bed net possession was high among the participants; however, its correct and consistent use was not assessed. The association between bed nets and malaria prevalence may be masked in some studies, as some patients get infective bites before retiring to bed or early morning due to the changing ecology and behaviour of the mosquito vector , impropePatient age is usually an important predictor in higher transmission settings, with infants under the age of 5 years being at a higher risk . HoweverThe effect of livestock at households on malaria transmission is dynamic, with the consensus that livestock can zoo-potentiate transmission when close to or inside human dwellings –59, but Culex mosquitoes was positive for Sindbis virus, in combination with a comparatively high infection rate with insect-specific flaviviruses [Plasmodium spp.-chikungunya dual infection has been shown to cause interferon gamma induced suppression of viraemia and joint pathology caused by arthralgic alphaviruses [This study attempted to determine acute arboviral and rickettsial infections in relation to malaria infection, as has been reported before in Kisumu County , Homa Baiviruses ; the lativiruses . On the aviruses . This isaviruses , and theaviruses .alphaviruses and flaviviruses are endemic [Rickettsia involve a combination of direct pathogen detection  and detection of a fourfold rise in antibody titers between acute- and convalescent-phase sera [Due to the low detection of arboviral infections by PCR, mostly because of a brief viraemic period and low viral load, previous studies in the same region have tried to highlight the circulation of arboviruses in human populations and/or determine their contribution to febrile illness by serology , 27, 71. endemic , 72. Thease sera , 74. In Amblyomma variegatum ticks with R. africae [versus microscopy and RDT as it highlights the advantages and limitations of each method.While previous studies have described high infection rates of  africae , the finRickettsia. While arboviruses or Rickettsia were not detected, they may be contributing a proportion of the febrile illness, at levels too low to be detected without very large studies. This presumption is on the backdrop of the reported circulation of arboviruses and Rickettsia by several serological studies in the same study region. The presence of livestock in the participants’ households is a variable of interest as reports on zoo-potentiation and zoo-prophylaxis are still conflicting. It is also an important factor in the control of arbovirus-transmitting mosquitoes, which are more zoophilic compared to malaria vectors. Distinguishing the proximity of the livestock to households is, therefore, of paramount importance in future studies. Only the ‘county of origin’ was a significant determinant of malaria infection in this study, highlighting the regions/counties of priority in malaria prevention programmes. This information is important in the implementation of targeted interventions.This study reports on the occurrence of malaria and associated risk factors in western Kenya in patients wherein it also sought to determine other causes of fever and related clinical features such as arboviruses and Additional file 1. Detailed description of primers and thermocycling conditions used for the detection of Rickettsia africae, arboviruses and Plasmodium spp.Additional file 2. Detailed description of the management of independent variables for statistical analysis.


Plasmodium falciparum infections prevail in low transmission settings, where immunity is expected to be minimal, suggesting an immune-independent effect on parasite densities. We aimed to describe parasite densities in pregnancy, and determine how gravidity and antibody-mediated immunity affect these, during a period of declining malaria transmission in southern Mozambique.Low-density P. falciparum infections at first antenatal care visits (n = 6471) between November 2016 and October 2019 in Ilha Josina (high-to-moderate transmission area), Manhiça (low transmission area), and Magude (pre-elimination area). Two-way interactions in mixed-effects regression models were used to assess gravidity-dependent differences in quantitative PCR-determined P. falciparum positivity rates (PfPRqPCR) and densities, in the relative proportion of detectable infections (pDi) with current diagnostic tests (≥ 100 parasites/μL) and in antimalarial antibodies.We documented PfPRqPCR declined from 28 to 13% in Ilha Josina and from 5–7 to 2% in Magude and Manhiça. In primigravidae, pDi was highest in Ilha Josina at the first study year (p = 0.048), which declined with falling PfPRqPCR , with no differences in antibody levels. Higher parasite densities in primigravidae from Ilha Josina during the first year were accompanied by a larger reduction of maternal hemoglobin levels , than in Magude  and Manhiça . In contrast, multigravidae during the transmission peak in Ilha Josina carried the lowest pDi (p = 0.049). As PfPRqPCR declined, geometric mean of parasite densities increased , and antibody levels declined among secundigravidae from Ilha Josina.The proportion of detectable and clinically relevant infections is the highest in primigravid women from high-to-moderate transmission settings and decreases with declining malaria. In contrast, the falling malaria trends are accompanied by increased parasite densities and reduced humoral immunity among secundigravidae. Factors other than acquired immunity thus emerge as potentially important for producing less detectable infections among primigravidae during marked declines in malaria transmission.The online version contains supplementary material available at 10.1186/s12916-022-02597-6. P. falciparum develops after repeated exposure, with a faster acquisition in high transmission areas ) with the participant’s consent. A brief form, including visit date, age, gravidity, gestational age based on the fundal height measurement, area of residence, and recent movements was completed. HIV status was recorded from the maternal health card. In case of an unavailable record, an HIV serological rapid test was done according to standard procedures for voluntary counseling and testing. Maternal hemoglobin levels were determined by HemoCue® Hb 201 + .P. falciparum in duplicate using real-time quantitative PCR (qPCR) assay targeting the 18S ribosomal RNA gene [DNA extracted from DBS was used for detection and quantification of RNA gene . ExperimRNA gene . The 101https://www.luminexcorp.com) [19]  , region om) [19] , full-le5 (PfRH2  and PfRH5 (PfRH2 )), and bexposure   and a random selection of approximately 2700 samples per year in both Magude and Manhiça would allow for the estimation of the 95% confidence interval (95%CI) of annual P. falciparum positivity rates between 20 and 5% in each of the 3 sites, with a margin of error lower than or equal to the expected positivity rate. Age was categorized as either younger than 18 years or 18 years and older, season as rainy (November to April) or dry (May to October) and year of recruitment from November to October in 2016–2017 as year 1, 2017–2018 as year 2,or 2018–2019 as year 3. Women reporting to have changed their residence area during their pregnancy, as well as women with missing information on gravidity, residence area or HIV, were dropped from the analysis. Detectable infections (Di) were defined as those with parasite densities equal or above 100 parasites/μL, which is the detection limit commonly used for standard RDTs [We estimated that the molecular detection of ard RDTs , and expt-test, respectively. Multilevel mixed-effects regression models with random intercept at site level were estimated to assess associations with qPCR positivity and pDi (logistic) and log-transformed parasite densities and mean fluorescence intensities (MFIs) as well as hemoglobin levels (linear). All models were estimated using transmission level (site) and gravidity (first pregnancy and two or more previous pregnancies) as independent variables and were adjusted for season, HIV status, residence in village or rural area, and place where molecular analysis was conducted (Mozambique or Spain). The hypothesis that the control mechanisms of P. falciparum infection in pregnancy are gravidity-dependent was assessed including an interaction term between gravidity and transmission levels (by site and period) in the models. Secondary analyses, which included genetic complexity of infections and secundigravidae, were assessed using linear and mixed-effects regression models, respectively. The significance level was set at 0.05. All analyses were performed using the Stata statistical program version 16 .Categorical and continuous data were compared between sites, gravidity groups, and time periods using Fisher’s exact test and Student Among the 10,439 first ANC visits recorded in the three antenatal clinics from November 2016 to November 2019, 8745 (8464 women) were included in the study. Not residing in the study area was the main reason for exclusion . On average, PfPRqPCR declined annually by 42%  in Ilha Josina (from 28% in year 1 to 13% in year 3), 44%  in Magude (from 5 to 2%), and 45%  in Manhiça  compared to Ilha Josina  and Magude , while no differences were observed among multigravidae nor during second and third years  out of the 6471 blood samples were positive for p = 0.004) and the proportion of detectable infections (p = 0.001) were higher among HIV-infected than HIV-uninfected women. Densities were 5.4-fold  higher in primigravidae than multigravidae (Table p < 0.001) than in Magude  and Manhiça . Forty-four percent (212/483) of the qPCR-detected infections were equal or above 100 parasites/μL and thus considered as infections detectable by conventional RDTs. Parasite densities , pDi was the highest among primigravidae in Ilha Josina (83%) compared to those in Magude (50%) and Manhiça  and tended to increase among multigravidae  or tended to increase , respectively, with no evidence of a modification by gravidity , P8 (p = 0.003), P1 (p = 0.029), and GEXP18 (p = 0.048) declined during the 3-year study period. Since P. falciparum infection was associated with increased antibody levels against all the antigens , HIV-infected , those residing in rural settings , and multigravidae , which was larger among primigravidae  than multigravidae . Among primigravidae, the largest reduction was observed in women from Ilha Josina , then Magude  and Manhiça , parasite densities and detectability, as well as the infection-associated reduction in hemoglobin, were higher among primigravidae from Ilha Josina, than those from Magude and Manhiça. Thereafter, parasite densities among primigravidae from Ilha Josina decreased as parasite rates dropped. This shift in parasite densities was not accompanied by changes in VAR2CSA-specific antibodies, which were similarly low among primigravid women in the three settings [Parasitemia in first pregnancy follows the opposite patterns of multigravid women, pointing to differences in the factors that determine parasite densities among women without pregnancy-specific immunity. When settings , as expesettings , 11). Itsettings  are alsosettings , 33. Thesettings .P. falciparum infections that can be detected with currently available diagnostic tools varies substantially depending on the intensity of transmission and gravidity. The proportion of detectable infections in primigravidae increased from 50 to 83% in areas with a PfPRqPCR of 4% and 28%, respectively. In contrast, detectable infections in multigravidae decreased from 44 to 21% with increasing parasite rates. Therefore, levels and trends of malaria burden, as well as fertility rates, are expected to affect the sensitivity to detect P. falciparum infections and as a consequence the efficiency of screen-based strategies [P. falciparum infections over periods in which malaria prevalence declines may increase among multigravid women as immunity wanes and parasite densities increase, but decrease among primigravidae as non-immune factors reduce parasite densities. Third, the results of this study highlight the value of monitoring malaria in pregnancy as an independent measure of malaria burden [The results of this study have several public health implications. First, the proportion of rategies , as wellrategies . Second,a burden , 34, 35 P. falciparum infections in the low transmission settings of Magude and Manhiça limited the ability to stratify women with two or more pregnancies in the analysis. Third, genotyping of parasite infections was not successful for low-density infections, which represents a large proportion of available samples. Fourth, age-dependent effects might have been attributed to pregnancy-specific immunity, given the correlation between age and gravidity. However, previous modeling data [This analysis has several limitations. First, inaccessibility of the placenta at booking visits prevents the direct study of parasite dynamics in that organ; however, parasite densities in peripheral and placental blood have been shown to follow similar trends , 29. Secing data  suggestsing data , 37. Fining data . HoweverP. falciparum infections in pregnancy, which is dependent on gravidity and changes in transmission intensity, affecting the sensitivity of current diagnostic tools as well as their role in malaria transmission. Finally, this study emphasizes the need to re-evaluate malaria conceptual frameworks, which are currently centered on the role of immunity, to better inform control and elimination strategies.Parasite densities in anti-VAR2CSA-naïve primigravidae decrease following a drastic malaria decline, independently of acquired immunity. This is in contrast with the reductions of immunity among secundigravidae which is accompanied by increasing parasite densities. The effect observed in first-time pregnant women, who have not yet developed pregnancy-specific immunity, might be explained by factors other than acquired immunity that favor low densities in low transmission areas. These observations challenge the dogma that all malaria infections progress to symptomatic infections as immunity wanes with a declining transmission. Both acquired immune and non-immune factors contribute to the substantial heterogeneity in the detectability of Additional file 1:Fig. S1. Map of Manhiça and Magude District in southern Mozambique where the study was conducted. Fig. S2: Performance of the qPCRs and quantitative suspension array assays along with the different experiments. Fig. S3. Study profile. Fig. S4. Genetic complexity of the infections by study area and period. Fig. S5. Antibody levels by studied factors.Additional file 2. Supplementary methods (Quantitative suspension array assay).Additional file 3. Table S1. Characteristics of study participants. Table S2. Interactions between centers and study variables on parasitological outcomes. Table S3. Interactions between gravidity and temporal trends on parasitological outcomes. Table S4.P. falciparum parasite rates, density and detectability between centers. Table S5. Annual changes in parasitological outcomes in pregnant women from Ilha Josina. Table S6. Annual changes in antibody levels by site and gravidity group.Additional file 4. Supplementary results .


Microscopy is still the gold standard for malaria diagnosis, and allows for species determination and quantitation, but requires trained microscopists, maintained microscopes and has detection limit issues. Consequently, there is a pressing need to develop and evaluate more sensitive and accurate diagnostic tests. To address this need we have developed a direct on blood mini PCR-NALFIA test that combines the benefits of molecular biology with low infrastructural requirements and extensive training.Rapid Diagnostic Tests (RDTs) have become the cornerstone for the management of malaria in many endemic settings, but their use is constrained for several reasons: (i) persistent malaria antigen  leading to false positive test results; (ii) This is a Phase 3 diagnostic evaluation in 5 African countries. Study sites  were selected to ensure wide geographical coverage of Africa and to address various malaria epidemiological contexts ranging from high transmission to near elimination settings with different clinical scenarios and diagnostic challenges. Study participants will be enrolled at the study health facilities after obtaining written informed consent. Diagnostic accuracy will be assessed following the WHO/TDR guidelines for the evaluation of diagnostics and reported according to STARD principles. Due to the lack of a 100% specific and sensitive standard diagnostic test for malaria, the sensitivity and specificity of the new test will be compared to the available diagnostic practices in place at the selected sites and to quantitative PCR as the reference test.This phase 3 study is designed to validate the clinical performance and feasibility of implementing a new diagnostic tool for the detection of malaria in real clinical settings. If successful, the proposed technology will improve the diagnosis of malaria. Enrolment started in November 2022 (Kenya) with assessment of long term outcome to be completed by 2023 at all recruitment sites.www.pactr.org) PACTR202202766889963 on 01/02/2022 and ISCRTN (www.isrctn.com/) ISRCTN13334317 on 22/02/2022.Pan African Clinical Trial Registry ( Plasmodium: P. falciparum, P. vivax, P. ovale, P. malariae and P. knowlesi. Malaria is a major global public health problem in tropical and sub-tropical regions with approximately half the world’s population at risk. In 2020, about 219 million malaria cases and nearly 450,000 deaths were reported worldwide. The majority of cases (92%) occurred in sub-Saharan Africa (SSA) [Plasmodium falciparum histidine-rich protein 2 (PfHRP2)-based RDTs, specific for Plasmodium falciparum only, and Plasmodium lactate dehydrogenase (pLDH)-based RDTs that detect all Plasmodium species, or combination tests with two test lines  [Malaria in humans is a life-threatening disease caused by single-cell (protozoan) parasites of the genus ca (SSA) . Most coPfHRP2 after successful treatment, which may lead to false positive test results [PfHRP2-based RDTs particularly in seasonal malaria transmission settings or under harsh environmental conditions [PfHRP2-RDT results increasingly reported from malaria endemic regions due to hrp2 deletions in the Plasmodium genome [P. falciparum infection is present. Finally, RDTs (pLDH-based in particular) have sensitivity and stability limitations, also resulting in false negative results [However, the implementation of RDTs is constrained by persisting  results –8. Furthnditions , 10. Of m genome –13. In t results , 14. Thi results , 16.Although malaria microscopy remains the standard for clinical diagnosis, the limit of detection (LoD) of malaria parasites by expert microscopy (which is as low as 10 parasites/μl) could miss very low parasitaemias. The LoD is highly dependent on the quality of the equipment, the microscopic reading technique, the experience of the reader and the daily workload .hrp2 deletions [In this context, there is a pressing need to develop more sensitive and accurate malaria diagnostic tests that circumvent the limitations of RDTs and microscopy. In general, molecular biology-based diagnostic tests are considered to be the most accurate methods for detecting malaria infections, irrespective of low parasite densities or eletions –20. Seveeletions . These peletions .s . This platform combines PCR with a nucleic acid lateral flow immunoassay (NALFIA) to detect Plasmodium species in a multiplex format [Plasmodium DNA in whole blood [To circumvent these challenges, the DIAGMAL consortium has developed a simple molecular diagnostic platform to diagnose malaria that can be used as a near patient testx format . NALFIA x format . Importale blood  by utilile blood , 23. RecThe main objective of the project is to evaluate the diagnostic performance of the mini-dbPCR-NALFIA test for malaria in field settings. To achieve this objective the following specific project objectives will be achieved through a blend of clinical, bio-medical and socio-economic research.Sudan); a seasonal malaria transmission setting where HRP2-RDT may have false positive outcomes (Burkina Faso); an area with possible HRP2-deletions and high prevalence of P. vivax malaria (Ethiopia); a low transmission (near elimination) setting (Namibia); and Kenya, which has several different malaria zones: meso-hyperendemic  in the lake and coastal areas; hypoendemic in the semi-arid regions, and potential for outbreaks in the highlands (non-immunes).To assess the diagnostic accuracy of the mini-dbPCR-NALFIA test for the diagnosis of malaria compared to routine diagnostic procedure(s) in place (microscopy and/or RDTs) and qPCR in different clinical and field settings: a high malaria incidence region .The study hypothesis is: “the mini-db-PCR-NALFIA will offer an improved diagnostic platform over microscopy and currently available HRP2-based RDTs for the detection of malaria infection in varied malaria epidemiological settings”.This will be a phase 3 diagnostic trial. Diagnostic performance will be assessed following the WHO/TDR guidelines for the evaluation of diagnostics  and the The research will be carried out in 5 African countries , each representing an epidemiology of malaria different from the other:Burkina Faso, the study will take place at the Clinical Research Unit of Nanoro (CRUN) trial site, located about 90 km from Ouagadougou. Malaria is hyper-endemic and transmission occurs throughout the year, with a peak during the rainy season (June–November). P. falciparum is responsible for >90% of all clinical malaria cases. Children under five years and pregnant women are the populations at highest risk. The total number of malaria cases for 2017 was >11 million. RDT remains the only diagnostic tool available to differentiate malaria from other infectious diseases in primary healthcare settings. The most common RDT used is PfHRP2. A high rate of false positive RDT is reported which leads to misdiagnosis. A recent study showed that almost 50% of other causes of fevers were diagnosed as malaria due to false positive RDT results [In Sudan is highly linked with climatic conditions. There are two transmission peaks; one during the rainy season (affecting most of Sudan) and the other during the winter season (notably in irrigation schemes and urban settings). In Sudan, 75% of the population is at risk. Malaria reported cases represent about 8.7% of total outpatient attendance and around 12.2% of hospital admissions. The main species is P. falciparum (87.6%). Diagnosis is made using microscopy and RDTs. The study will be performed in Gezira State under the supervision of the Blue Nile National Institute for Communicable Diseases (BNNICD)–University of Gezira in the outpatient clinics of three teaching hospitals and health centres with whom BNNICD has previously been conducting malaria research. These study sites may recruit more than 2,000 malaria cases per year.Malaria transmission in Ethiopia will take place in Arba Minch , Melka Werer (Afar) and Maksegnit (Gondar Zuria District). Arba Minch town  is located 505 km south of Addis Ababa in Gamo Gofa zone with an average elevation of 1,209 m above sea level, an average annual temperature is 29.7°C and rainfall of 700 mm. Malaria is seasonal (September to December) and occurs in epidemic type where both P. falciparum and P. vivax are prevalent. There are two public health centres and one public hospital in Arba Minch town that provide healthcare services for the community in the town and surrounding areas. Around 179,386 febrile cases were attended to at these public healthcare facilities in Arba Minch in 2020 (MoH Report).P. falciparum and P. vivax malaria are endemic.Maksegnit town (Gondar Zuria District) has one health centre and a malaria research unit. Gondar Zaria District is in North Gondar Zone, Amhara National Regional State of Ethiopia. Maksegnit is located at an altitude ranging between 1,107−3,022 meters above sea level, temperature ranging between 14–20°C  and annual rainfall ranging between 1,030–1,223 mm . Both Melka Werer (Amibara District) is located 265 km northeast of Addis Ababa in the Middle Awash valley. The climate of Amibara is generally semi-arid with temperatures ranging from 25°C−35°C and with an average annual rainfall below 600 mm. The altitude of Amibara lies between 720−1100 m above sea level. The 2019 population projection for Amibara District was 102,327. In Amibara District, there is one hospital , three health centers , 10 private clinics, and 17 health posts that provide health services to the population. Malaria is the top leading cause of morbidity in Amibara District. About 386,096 febrile illnesses and 15,0159 malaria cases were reported in 2020 by the health facilities.The study in Kenya, the study will be performed in two sites: one at the Lake Region (Busia County) which is endemic for malaria, and one in Kajiado County, a seasonal malaria area. At both sites 80% of the population are at risk; all malaria species occur but P. falciparum accounts for 98% of cases. Transmission is highest in May–July and January–March during the two rainy seasons. On a typical day, 50–100 patients with suspected malaria may be seen in a single health facility. Microscopy is the primary diagnostic approach in health facilities with laboratories, with RDTs also used for emergencies.In Namibia, the study will be conducted in an area that is classified as near elimination, the Zambezi region (North Eastern Namibia). The Zambezi region is primarily rural with most of the population engaged in subsistence farming. Malaria transmission is very heterogeneous in Namibia with transmission occurring during the rainy season peaking between January and May. The Zambezi region has accounted for more than 70% of the country’s malaria cases since 2020, the country at large had an incidence of 1.16 cases per 1000 in 2019. The country aims to eliminate malaria by 2022 and conducts case investigation of all malaria cases using Reactive Case Detection as malaria cases (both secondary and asymptomatic) have been shown to cluster around index cases. Reported malaria cases are almost all due to Plasmodium falciparum.In This selection of study sites also ensures a geographical coverage of Africa with partners from western, eastern and southern regions.For Sudan, Ethiopia, Burkina Faso and Kenya, the study will recruit participants who fulfil the following inclusion criteria:Patients with clinically suspected malaria of all ages presenting at the health facility;Coming from the health centre catchment area;Informed consent from patients, or parents or guardians (in case of minors).For Namibia, the study will recruit participants who fulfil the following criteria:Residents of one of the selected households or have slept the night before in this household.Informed consent from individual participants.The exclusion criteria are not meeting the above criteria, or parents or guardians (in case of minors).Written informed consent will be obtained from all the study participants  before enrolment. After an interview with the study clinician community health worker or other qualified member of the study team, the participant  will be asked to document their consent by signing an informed consent form; if the patient is a minor , the consent must be given by a parent or legal guardian. For minors in a specific age range informed assent is also obtained. Signed informed consent (or thumb-print whenever the participant or parents/guardians are illiterate) must be obtained before any blood sample or tests related to the study are carried out.In addition, consent for the cost effectiveness study will be sought at the same time as enrolment to the diagnostic trial. Written informed consent will also be sought ahead of all structured interviews with key stakeholders for the health systems analysis.In the informed consenting procedure, participants and parent/guardians will be informed about future investigations on the collected blood samples. They will be informed that future investigations will only focus on understanding the performance and quality of the diagnostic procedures under study. For this purpose, part of the collected biological specimens may be transported and tested in Amsterdam (The Netherlands). No genetic testing about the human host will be performed. Some patient data will also be used for the health economics evaluation.The diagnostic performance of the mini-dbPCR-NALFIA will be compared to the standard malaria diagnostic practices in place in the 5 participating countries (i.e. microscopy and/or rapid diagnostic test). Established qPCR will be used as the reference standard.Depending on the routine diagnostic procedures in place at each site, a rapid diagnostic test (RDT) or microscopy, or both, will be performed by the resident staff. RDT will be performed according to the manufacturers’ procedures. Thick and thin blood films will be prepared and Giemsa stained. Blood slides will be independently read by two expert microscopists. Parasite density will be calculated by counting the number of asexual parasites per 500 leucocytes in the thick blood film, based on an assumed WBC of 8,000 /μl by light microscopy at 1000 x magnification. In addition to routine diagnostic procedures in place, an additional blood sample  will be collected for test evaluation (index and reference tests). Blood samples will be properly labelled with participants’ initials, inclusion number, protocol number, the study day and the date the sample is taken. For the index test (mini dbPCR-NALFIA) blood will be collected into an EDTA tube. For reference testing (qPCR), three blood spots will be collected on Whatman 3 filter paper ; one samOnly a single additional blood sample will be collected for the study. Treatment decisions will be made on the basis of routine diagnostic procedures in place. There will be no interference with standard diagnostic and treatment practices. There is therefore no need to define criteria for discontinuing or modifying interventions.This study requires a single sampling point and there is no need for cases to be subjected to further interventions. Consequently, there is no need to design strategies to improve adherence to interventions.Not applicable, as this is a diagnostics study and only a single blood sample per study case will be tested. Prior use of anti-malarial drugs might influence diagnostic test performance, and prior drug use in the 14 days preceding collection of the blood sample will be recorded.As this is a diagnostic trial in which we are not testing any drug, such visits are not absolutely necessary. However, for ethical and safety reasons we will make a reasonable effort to contact all patients to check their health status and make sure they are recovering well. This visit can be done either physically or by telephone contact. Those who are not recovering well will be asked to return to the health facility for assessment and further management by the health facility staff and this action will be recorded in the CRF.Outcomes will be reported according to STARD principles . For eacParticipants will be asked to provide a clinical specimen and some demographic data on the day of enrolment. No further assessments or visits are planned in the context of this studyThe sample size for each country has been calculated according to the expected disease prevalence of the field sites to give adequate statistical definition to test sensitivity and specificity , 25 SampSample size calculations are further based on a conservative estimation of the number of actual cases amongst febrile patients  attending the various health facilities participating in the study. Furthermore, we have taken into account that there might be a withdrawal of consent of around 20% in each setting (most likely less). Based on these assumptions the following sample sizes have been determined for the following countries :Sudan (10% incidence): 876 study cases;Ethiopia (10% incidence): 876 study cases;Burkina Faso (20% incidence): 438 study cases;Kenya (25% incidence): 351 study cases;Namibia: prevalence is <1.5% in the near elimination study region. prevalence is <1.5% in the near elimination study region. Therefore, participants will be recruited using passive and reactive case detection (RACD) targeting 2,000 people in the study areas as was also done in a previous study .Prior to the start of the study, in some sites, communities and community leaders will be informed about the purpose of the study to enhance participation. All study sites have a sufficiently large population and malaria incidence to reach the projected sample size.Not applicable, as there will be no allocation to different study arms. All subjects will be subjected to the same procedure; i.e. collection of a blood sample for diagnostic testing.Not applicable, as there will be no allocation to different study arms. All cases will receive the same intervention.Not applicable, as there will be no allocation to different study arms. All cases will receive the same intervention.There is no procedure for blinding as all participants will be following the same study procedure. Study staff executing the different diagnostic tests will be blinded from the results of other tests (.i.e. tests that they are not performing). The results of routine diagnostic testing in place at the study site will be used to make treatment decisions as per guidelines of the respective national Malaria Control Programmes.There is no procedure for unblinding.Study staff will be adequately trained in execution of diagnostic tests performed in the framework of the study, data collection and data recording prior to the start of the trial. All data must be initially documented in the source documents, and then entered in the Case Record Form. For this study, an electronic database (developed on Open Clinica Software) will be used, which can be defined and entered remotely using the Internet. The CRF and database will allow identification of the study, site and patient; recording of the selection and inclusion of patients in the study; and recording of all data collected at each different study site.A singe sample collection is foreseen with no follow-up. Therefore, there is no need to develop a plan to promote participant retention or to complete follow-up.Data management will be handled by the Institut de Recherche en Sciences de la Santé (IRSS) which has the required expertise in all components of data management of clinical trials and epidemiological studies. The data management office is located in the main campus of the Clinical Research Unit of Nanoro (CRUN). Data will be collected onto paper-based source documents before being captured into the electronic CRF developed in Open Clinica. Data will be entered online in all five sites and sent to the central server located at CRUN. The central data base will be managed at the CRUN by a data manager, who will run regular consistency checks and identify queries to be resolved by the local investigators in each country. This system will allow the rapid identification of potential problems. Data entry and review will be performed following the Data Entry Guidelines and the Data Management plan provided to the sites by the CRUN. Besides this central management, as mentioned above, a study monitor will visit each site at least once and check the information entered into the electronic CRF against the source documents available on site. Any modification made on the electronic CRF will be automatically registered. The final database will be obtained after resolution of all queries and will be locked for statistical analysis to be carried out according to a pre-established plan that will be developed by the study statistician in collaboration with the site investigators. The statistical analysis plan will specify how to deal with statistical issues such as missing data and protocol violations.Every possible measure will be taken to ensure that all personal data collected during the study will be appropriately protected. The collection of personal information will be restricted to meet the objectives of the project, and only data relevant for the execution of the project and interpretation of data will be collected. Collected information will be kept for a minimal time period, which is regulated by national or international law. All personal data collected during the study will be kept confidential. A numerical identifier and participants’ 3 initials will be assigned to each CFR and sample. None of the records contain individual name identification. All signed informed consent and assent forms and other personalised information will be kept under lock and key and will be available only to the project coordinator, principal investigators and data managers. Information stored in electronic databases will be protected by unique usernames and passwords, which will only be made available to appropriate authorised personnel. All records and data entry will be checked independently on-site by the research clinician and/or by the local PI to ensure accuracy. The collected information will never be transferred to countries lacking appropriate data protection or made available without restriction to third parties that are not directly linked to the project.Plasmodium species only. There will be no analysis of human genetic material. Collected specimens could be used for future diagnostic test development and evaluation, and if this will be the case, explicit informed consent from study participants will be obtained.Blood samples will be collected for routine malaria diagnostic tests and molecular analysis with PCR, which is used as the reference test for the diagnostic procedures followed in this trial. This work will be restricted to the assessment of the presence of The outcomes of the evaluations will be analysed using 2 x 2 tables  and comparisons will be made with standard diagnostic procedures in place (microscopy and/or RDT) and qPCR as the accepted reference test to determine agreement between different tests . Positive and negative predictive values will be calculated. Sensitivity and specificity of the db-PCR-NALFIA are assumed to be 95%.None planned.P. falciparum detection compared to routine diagnostic practices for malaria in the 5 different study localities .Next to the assessment of the diagnostic performance of the molecular platform, an economic evaluation and health systems analysis will be conducted to identify the incremental costs and benefits to healthcare providers and users of introducing and routinely using the mini-dbPCR-NALFIA for The performance of the mini-dbPCR-NALFIA in terms of sensitivity and specificity will be combined with costs to the health system  to estimate the cost-effectiveness of the novel malaria diagnostic tool. The health outcomes will build on the Phase 3 trial primary endpoints and model the cost per disability-adjusted life years averted.Potential costs, barriers and facilitators associated with the implementation of the mini-dbPCR-NALFIA will be identified from both the supply and demand side and will imply some data collection alongside the main trial.Supply Side—health system information on barriers and facilitators will include:Introduction / Start-up costs: Information will be gathered through document reviews and during key informant interviews at different health system levels  in order to understand which activities should be undertaken for the introduction of the mini-dbPCR-NALFIA and their associated resources and expected costs at each level of the health system  In response to some of the key learnings of the recent Lancet Commission on Diagnostics , we willImplementation / Running costs: Interviews will be conducted with key personnel at health facilities where mini-dbPCR-NALFIA will be used as part of the main study to help ascertain the costs and resources needed to routinely use dbPCR-NALFIA. These costs will largely reflect the cost of the tool and associated personnel costs for administration.Feasibility & Scale up: If the mini-dbPCR-NALFIA is cost effective, is it also affordable? How can the supply chain best accommodate this novel diagnostic tool? What would be the market barriers? What is the potential scope for private sector involvement in any scale up? This information will be gathered through document reviews and during key informant interviews in each of the five countries conducting the trial.Demand side—recipients’ barriers/facilitators will be identified by collecting information from participants involved in the trial on:Costs of seeking diagnosis for fever or other symptoms suggestive of malaria .To understand the potential demand for a new product that is yet to reach the market, such as mini-dbPCR-NALFIA, we will ask patients their willingness to pay (WTP) for the product. WTP is a common economic approach that quantifies the hypothetical value potential users attribute to a product and its various characteristics, and the benefit they expect to obtain from it. Participants enrolled in the trial will be asked to participate in an economic method called the bidding game where they will be asked if they are willing to pay a base amount of money that will go up or down depending on the participants reply at each bid.The perception of risk from malaria may impact on the uptake of the mini-dbPCR-NALFIA and have implications for delivery strategies (e.g. facility-based versus community-based screening). A survey divided into perceptions of local health problems, malaria knowledge, prevention measures, and malaria risk perception will explore how study participants view the threat of malaria and how this may influence their perceptions of the near POC being trialled.Protocol non-adherence is not expected to occur as the trial only involves a single collection of a blood sample from the study cases. In case missing data occur, the whole data set of a participant will be removed from the analysis. This removal will be compensated by a slight over recruitment in number of study participants.The study has been registered in two accessible clinical trial registers and the information contained in these registers provide sufficient insights into the full trial design. The final results of the study will be published in peer-reviewed Open Access journals and presented at scientific meetings. None of the trial material may be disclosed to any party not directly involved in the study without written permission from the consortium partners. Presentation and publication of the trial results will be jointly carried out by the project investigators. Each African collaborator will be in charge of sharing relevant results with their respective health authorities, including the National Malaria Control Programmes. This will be done through ad-hoc meetings, data dissemination workshops and/or during Malaria Days in each country. Trial data will be accessible for inspection by appropriate health and regulatory authoritiesThe trial is coordinated by dedicated trial staff at CRUN  and includes data management. CRUN is responsible for generic study protocol development and implementation. Each country is responsible for the implementation of the protocol in its respective sites, ethics review of the study protocol and other licensing requirements. An independent trial monitor has been appointed who will perform monitoring visits to all 5 study countries.CRUN (Burkina Faso) is responsible for centralised data management and monitoring. The central data base will be managed at CRUN by a dedicated data manager, who will run regular consistency checks and produce queries to be resolved by the local investigators. This system will allow the rapid identification of potential problems. Data entry and review will be performed following the Data Management Plan. Data management is independent from the sponsor.The intervention is unlikely to result in (unexpected) adverse events or harms. An incidental findings policy is in place. This project will not involve research conducted towards genes and/or the genetic basis of diseases on human subjects. The research is not intended to provide subjects or their families with specific information about their genetic status. Furthermore, the research will be focused on the diagnosis of malaria only (a very common and well known condition in the study sites) and not on any other diseases or conditions, and does not actively search for human genetic disorders. Therefore, any other medical problems detected in patients with malaria or those testing negative for malaria will be managed appropriately within the health system. The risk that incidental findings occur in this study is very low though it might be the case when blood slides are examined by microscopy that there is a chance that other parasites, such as trypanosomes, may be found. In such cases the attending clinician will be informed to undertake appropriate medical actions.Each site will be visited by an independent monitor at least once during the conduct of the trial plus a study initiation visit at the start of study activities and a close-out visit after the last patient has completed the study procedures. The task of the Monitor is to verify the best conduct of the study through frequent contacts by telephone and in person with the Principal Investigator and site staff, in accordance with applicable regulations, Good Clinical Practice requirements and study-specific Standard Operating Procedures. The objectives and specific tasks of the Monitor are described in the ICH Guidelines E6. Source documents will be kept for each participant in the study. All information in the electronic CRF must be traceable to these source documents in each participant’s file. The Monitor, who is bound by a confidentiality agreement to protect participants’ confidentiality, has access to all relevant source documents to confirm their consistency with the electronic CRF entries.Protocol amendments will be communicated to the funder (EDCTP), sponsor (AMC) and relevant ethics review bodies in the country concerned.Dissemination of the study findings is planned through scientific papers and conference attendance, and also through meetings with various audiences of interest, including key stakeholders such as the health facilities involved, public health authorities and diagnostic companies who might be interested in bringing the diagnostic platform to market.s [Plasmodium infections, but requires qualified laboratory technicians, well maintained microscopes and a conventional laboratory. Microscopy also has a detection limit for low density infections and it is not accessible to many patients in endemic areas. This is even truer for diagnostics based on molecular biology, the most sensitive tools for the diagnosis of malaria, which needs specific equipment, sophisticated infrastructure and high-level trained laboratory personnel [Plasmodium species in a multiplex format [Among the factors that have contributed to the introduction of malaria RDTs into the health system in LMICs are the short running time, easy read-out system, limited infrastructural needs combined with their relatively low costs , 30. Unfs –34. Malaersonnel . To circx format .Plasmodium species in real life conditions. Based on previous studies [hrp2 deletion in P. falciparum leads to false negative results, or settings where sensitive diagnostics are needed to detect the last reservoir of parasite infection to achieve malaria elimination [This study is designed towards the validation of the clinical performance of the mini-dbPCR-NALFIA for the detection of  studies , 22, 23,mination , 16, 36.This evidence will be complemented with additional data on the costs associated with implementation, and information on patients and healthcare providers’ acceptance of the new diagnostic device. These activities will be implemented in parallel with the assessment of the new diagnostic platform. Despite successful evaluation of the diagnostic test, its integration within the health system may be compromised by patients’ and/or health staff’s low acceptability of the platform , 38. ThiOne of the major expected key deliverables of this project is to pave the route to marketing of the platform. Indeed, RDTs and microscopy will continue to be the backbone of clinical diagnosis in the near future, but performance limitations of RDTs mean that they cannot be used to screen populations for malaria infection in low transmission settings, for surveillance of populations as part of elimination programmes, in settings where RDTs have low specificity such as for treatment follow up, or in regions where HRP2-deleted parasites are becoming increasingly prevalent. It is into these market niches that the mini dbPCR-NALFIA fits very comfortably. For example, there are currently 18 countries with malaria elimination or eradication programmes in place. While these programmes vary in the number of tests carried out per year, the potential market size is estimated at millions of tests per year. The market for a sensitive screening diagnostic test will increase in future years as more countries introduce elimination and eradication programmes or will have measures in place to sustain their malaria free status.The project may also face some operational challenges. Currently many research resources are allocated to combat the worldwide Covid-19 pandemic. Fortunately, our research sites are highly dedicated to perform malaria research, as this important infectious disease is still considered to have a huge impact on human health in the coming years. This is stressed by the fact that vaccines are not readily available and control measures are starting to lose their effectiveness due to emerging resistance to commonly used anti-malarial drugs. Furthermore, civil unrest is occurring in some of the participating countries. However, so far these have not affected our studies.This is version 1.2, dated 2 July 2020, of the protocol. Ethics approvals have been obtained. Study staff are recruited and trained. At the time of submission, the recruitment of study participants started on 10 November 2021 in Kenya, on 7 December 2021 in Burkina Faso, 4 January 2022 in Sudan and 23 February 2022 in Namibia. Start in Ethiopia is pending. The trial is ongoing. Recruitment is expected to be completed 31 December 2022 and the final results of the study are expected end of 2023.S1 File(DOCX)Click here for additional data file.S2 File(DOCX)Click here for additional data file. 28 Jul 2022Phase 3 evaluation of an innovative simple molecular test for the diagnosis of malaria in different endemic and health settings in sub-SaharaN Africa (DIAGMAL)PONE-D-22-16998Dear Dr. Schallig,We’re pleased to inform you that your manuscript has been judged scientifically suitable for publication and will be formally accepted for publication once it meets all outstanding technical requirements.Within one week, you’ll receive an e-mail detailing the required amendments. When these have been addressed, you’ll receive a formal acceptance letter and your manuscript will be scheduled for publication.http://www.editorialmanager.com/pone/, click the 'Update My Information' link at the top of the page, and double check that your user information is up-to-date. If you have any billing related questions, please contact our Author Billing department directly at authorbilling@plos.org.An invoice for payment will follow shortly after the formal acceptance. To ensure an efficient process, please log into Editorial Manager at onepress@plos.org.If your institution or institutions have a press office, please notify them about your upcoming paper to help maximize its impact. If they’ll be preparing press materials, please inform our press team as soon as possible -- no later than 48 hours after receiving the formal acceptance. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information, please contact Kind regards,Lucinda Shen, MScStaff EditorPLOS ONEJournal requirements:When submitting your revision, we need you to address these additional requirements.1. Please ensure that your manuscript meets PLOS ONE's style requirements, including those for file naming. The PLOS ONE style templates can be found at https://journals.plos.org/plosone/s/file?id=wjVg/PLOSOne_formatting_sample_main_body.pdf and https://journals.plos.org/plosone/s/file?id=ba62/PLOSOne_formatting_sample_title_authors_affiliations.pdf2. Your ethics statement should only appear in the Methods section of your manuscript. If your ethics statement is written in any section besides the Methods, please move it to the Methods section and delete it from any other section. Please ensure that your ethics statement is included in your manuscript, as the ethics statement entered into the online submission form will not be published alongside your manuscript.http://journals.plos.org/plosone/s/supporting-information.3. Please include captions for your Supporting Information files at the end of your manuscript, and update any in-text citations to match accordingly. Please see our Supporting Information guidelines for more information: Reviewers' comments: 22 Aug 2022PONE-D-22-16998 Phase 3 evaluation of an innovative simple molecular test for the diagnosis of malaria in different endemic and health settings in sub-SaharaN Africa (DIAGMAL) Dear Dr. Schallig:I'm pleased to inform you that your manuscript has been deemed suitable for publication in PLOS ONE. Congratulations! Your manuscript is now with our production department. onepress@plos.org.If your institution or institutions have a press office, please let them know about your upcoming paper now to help maximize its impact. If they'll be preparing press materials, please inform our press team within the next 48 hours. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information please contact plosone@plos.org. If we can help with anything else, please email us at Thank you for submitting your work to PLOS ONE and supporting open access. Kind regards, PLOS ONE Editorial Office Staffon behalf ofMiss Lucinda Shen Staff EditorPLOS ONE


Our robust strategy that identifies and quantifies malaria can play a pivotal role in disease control strategies.Eradication of malaria, a mosquito-borne parasitic disease that hijacks human red blood cells, is a global priority. Microscopy remains the gold standard hallmark for diagnosis and estimation of parasitemia for malaria, to date. However, this approach is time-consuming and requires much expertise especially in malaria-endemic countries or in areas with low-density malaria infection. Thus, there is a need for accurate malaria diagnosis/parasitemia estimation with standardized, fast, and more reliable methods. To this end, we performed a proof-of-concept study using the automated imaging (NanoZoomer) platform to detect the malarial parasite in infected blood. The approach can be used as a steppingstone for malaria diagnosis and parasitemia estimation. Additionally, we created an algorithm (ParasiteMacro) compatible with free online imaging software (ImageJ) that can be used with low magnification objectives  both in the NanoZoomer and routine microscope. The novel approach to estimate malarial parasitemia based on modern technologies compared to manual light microscopy demonstrated 100% sensitivity, 87% specificity, a 100% negative predictive value (NPV) and a 93% positive predictive value (PPV). The manual and automated malaria counts showed a good Pearson correlation for low- (R Plasmodium-infected female Anopheles mosquito takes a blood meal from the host, depositing sporozoites into the skin and bloodstream which then migrate to the liver and transform into merozoites. Subsequently, the merozoites are released which invade red blood cells (RBCs), and transform into ring, then trophozoite and finally schizont stages. The schizont then egress from the cell, releasing merozoites that invade new uninfected RBCs, reinitiating the erythrocytic cycle [Malaria is one of the deadliest parasitic infections among humans, especially in tropical regions. Symptoms of the disease include fever, vomiting, headaches, and in severe cases, may result in death . Althougic cycle .One of the factors that has contributed towards the persistence of malaria is the scarcity for routine use of automated standardized/optimized diagnostic tools that allow for accurate detection and parasite density estimation in asymptomatic individuals ,5,6. ManPlasmodium spp. are currently identified with light microscopy, rapid diagnostic tests, and molecular detection assays , each having their own drawbacks. The standard laboratory diagnosis procedure involves identifying the Plasmodium spp. through light microscopy, which remains the gold standard method. However, this approach is time-consuming, cumbersome, and error-prone with different caveats at each step starting from sample preparation to visualization of the parasites. Light microscopy has certain limitations due to a poor-quality control system with the possibility of misdiagnosis especially in cases with low parasitemia or mixed infections [Plasmodium species [fections ,12,13,14fections . The prifections ,17, and  species . The sen species ,20. Howe species . MoreoveThe NanoZoomer HT Scan system 2.0  is a slide scanner that is suitable for high-throughput applications with high sensitivity, and high resolution. The salient features of the NanoZoomer include, scanning capability of standard-size (26 mm × 76 mm) slides, the slide loader can handle upto 210 slides at a time/per batch, it is fully automatic without the need of an attendant, wide range of software solutions and Z-stack feature. The system has the capacity to scan the area of 1 cm × 1 cm in 1 min . The sysLiterature search shows that the diagnostic accuracy of malaria by any method relies on many factors and can be adversely affected by the inter-examiner variability, observer bias, as well as human errors imposed by large-scale diagnoses in resource-constrained areas of endemic regions . To overAll reagents used in the study were research grade. A+ blood was purchased from Bio IVT . Parasite strains (HB3 and 7G8) were obtained from BEI Resources  .P. falciparum) were cultivated at 1% hematocrit in RBCs in Roswell Park Memorial Institute 1640 medium (RPMI 1640) [2-5% O2. Media was changed every 6 h and growth of the parasites were monitored by Giemsa-staining [Parasites (MI 1640)  supplemestaining . Both HBstaining  of GiemsFor automated counting, malaria thin blood smear slides were digitized using the NanoZoomer HT Scan system 2.0  . The imaGiemsa-stained thin blood smear slides were collected and photographed at the Pathology Core, Wellman Center for Photomedicine, Massachusetts General Hospital using the NanoZoomer   and lighParticle size (area) and the degree of roundness were additional inclusion variables. ImageJ preprocessing and particle analysis commands provided a preliminary particle count, and the software plotted the particle size-frequency distributions. The ImageJ macro was recorded from within the opensource ImageJ software and was enhanced to accurately find and count infected and non-infected RBCs based on their difference in appearance and associated pixel value. Infected RBCs were visible in blue due to the stain (Giemsa) targeting hemozoin crystals present in them. Blue has a lower pixel value compared to uninfected RBCs.Briefly, the code first used the “make binary” function to divide the structures in the image into two-pixel values 0 and 255. Then, the function “set measurements” removed the background by excluding all the high pixel values showing the remaining hemozoin particles. The “analyzed particle” function assigned regions of interest (ROI) to the hemozoin particles, counted by the following “for loop” function. The hemozoin crystals’ ROIs were then tracked in a separate image in two separate stacks, allowing the user to control for accurate crystal inclusion manually.In the second section of the code, the original image was re-opened, and pixel value thresholds were set to distinguish between infected and non-infected RBCs. A size inclusion threshold was set to reduce included artifacts by the “analyzed particles”. The macro repeated the conversion into a binary mask to find the particles. The “fill holes” component was used to create homogenous particles to aid the “analysis particle” function. The “watershed” function then roughly separated aggregated particles. The “analyze particle” function assigned ROIs to the found particles, and the macro opened the original RGB image with the overlaid ROIs, which allowed for manual graphic interpretation. The “for loop” function counted either red or blue cells based on the set pixel value thresholds. The final portion of the code calculated the hemozoin crystals, the total RBCs and estimated parasitemia in the image and “printed” the values in the results window.Pearson correlation coefficient was calculated by a two-tailed analysis using Graphpad Prism (9.0).Both HB3 and 7G8 parasite strains were successfully cultured, after which the parasites were diluted to varying parasitemia levels. The level of parasitemia was estimated by Giemsa-stained thin blood smears that gave parasite levels ranging from 4.8–7.9% for HB3 and 0.15–0.89% for 7G8 strains. To validate the high-throughput NanoZoomer detection system for detecting malaria and evaluating parasitemia, images were taken from slides prepared from the parasite cultures. These images were imported to a computer and the algorithm (ParasiteMacro) was run to estimate parasitemia . A totalp), False positive (Fp), True negative (Tn), False negative (Fn).The results obtained from conventional manual microscopy were considered as true and were compared to the automated malaria detection/parasitemia estimation system (NanoZoomer/ParasiteMacro). The system showed 100% sensitivity, 87% specificity, a 100% negative predictive value (NPV), and a 93% positive predictive value (PPV) . The equP. falciparum varying infections between the automated versus the manual malaria counts for both high and low parasitemia levels by the scatter plot analysis. The automated and manual counts showed a good Pearson correlation  for the high as well as the low parasitemia   .The automated parasitemia estimates were recorded and compared to the corresponding manually established counterparts for HB3 , 7G8 Ta and non-P. falciparum strains (HB3 and 7G8) at high and low parasitemia, respectively. HB3 is chloroquine sensitive whereas 7G8 is chloroquine resistant. Both strains have different biological characteristics and their inclusion in the study has further strengthened our conclusions on the feasibility of using our novel approach for detection of malaria. To this end, we found our approach to be cumulatively 100% sensitive and the specificity was 87%. The reduced specificity was due to false positives when using the 5× or 10x objectives. This could be due to overstaining of the slides or poor washing after Giemsa stain. The NPV and PPV were calculated as 100% and 93%, respectively.In the present study, we have developed a machine learning platform to make malaria detection robust. Our algorithm (ParasiteMacro) is compatible with online public access software using low power objectives in automated  and manual  imaging systems. We developed a Java code, and its application helps in the fast analysis of malaria. To test our system in this study we used two different An expert microscopist takes an average of 15–30 minutes to diagnose malaria and estimate the level of parasitemia in a thin blood smear Giemsa-stained slide ,33. ThisSmartphones have been used for malaria detection, especially in resource-poor environments ,39,40. FPlasmodium species but also estimate Plasmodium associated parasitemia levels . Our novel approach is easy to use with no steep learning curve. It is recommended that the application of detection and diagnostic devices be focused on the simplicity of an assay to be implemented with minimum infrastructural requirements, such as a reliable supply of electricity in the area [Plasmodium species with minimum requirements such as an inexpensive microscope and a computer. The new technique considerably improves the sensitivity of malaria diagnosis. Moreover, the method is proposed to be implemented towards a clinical diagnostic accuracy trial to determine the feasibility of use in clinical cases. We thus hypothesize that this robust methodology can play a pivotal role in informed decision making in busy settings. The code will increase the precision of the calculated reservoir size of infections with low- and high-density parasites influencing malaria epidemiology. The identification of 100% positive samples in this study indicates that this method may be an appropriate survey tool to estimate the amount of the asymptomatic low-density infections accurately. This could also improve the efficacy of mass screening and treatment (MSAT) approaches in low incidence settings, given its improved performance. The method described here can be used as an early steppingstone into malaria detection and parasitemia estimation. However, there are some limitations of our study, i.e., the performance of the system was only validated by in house varying parasitemia cultures and no patient samples were analyzed. The quality of the Giemsa-stained blood smears plays a vital role towards getting quality results. We also did not test the system for identifying the different life stages of the parasite. The technique was only compared to manual malaria counts and should be compared with real-time PCR and flowcytometry, etc. The results should be interpreted carefully as other intraerythrocytic parasites, malformed RBCs, cellular components such as WBCs and platelets can also be stained with Giemsa. Finally, our system was not tested to differentiate various Plasmodium species ; instead, only P. falciparum was used as the model organism for malaria. Future studies focusing on comparing the sensitivity and specificity of other malarial detection methods under field conditions should be done using this technology. However, this would be challenging as in field conditions blood smears are often dirty and contaminated, nevertheless the results presented in our study warrant further investigation of the novel platform.In this proof-of-concept study, we show that the NanoZoomer, combined with the Java code (ParasiteMacro), can be widely used to not only identify the area . The metThe novel method described here using a new Java code algorithm (ParasiteMacro) offers a new machine learning approach with a NanoZoomer-automated framework. In specific cases, such as prevalence estimation, reactive case detection, or MSAT, it is now essential to use robust technologies. Employing our machine learning algorithm to automated detection (NanoZoomer) systems of malaria would allow detection in low density and high prevalence areas. Taken together this platform will help in the eradication of malaria. The next steps would be to test this approach on clinical samples and compare it with other methodologies.