The Ministry of Health, Ghana, in accordance with global policy, recommends that all suspected malaria cases be confirmed parasitologically before treatment. Not all clinicians, however, base their treatment on test results. Patients also spend a lot of time at health facilities waiting to consult a clinician before being asked to go for testing and to see a clinician with test results. The purpose of the study was to determine if testing all children aged 6 to 59 months with fever reporting at an outpatients department (OPD) for malaria before consultation with a clinician (pre-consultation testing) will influence clinicians to adhere to test results and also reduce the time spent by such patients.A quasi-experimental study design was used involving two randomly selected government-owned hospitals in the Northern Volta, Ghana. In each hospital, 439 children were recruited between November 2018 and January 2019. The intervention hospital implemented pre-consultation testing. In the comparator arm, standard practices, which involved patients seeing the clinician before he/she decides whether to send the patient for testing or not, were maintained.Out of 878 children screened the overall prevalence of malaria was 31.9% by malaria rapid diagnostic test (RDT) and 26.7% by microscopy. Clinicians in the intervention arm adhered more to the malaria test results than those in the comparator arm . The proportion of children who tested negative but were still diagnosed with malaria was significantly lower in the intervention arm compared to the comparator arm (8.4 vs. 21.2%: p < 0.001). Clinicians and mothers/caregivers in both arms preferred pre-consulting testing. Six out of every 10 mothers/caregivers in the comparator arm viewed the waiting time as ‘too long’’ compared to 4 out of every 10 mothers in the intervention arm. On average, patient waiting time was significantly lower in the intervention arm (2.61 h) than in the comparator arm (3.42 h).Pre-consultation testing significantly improves clinicians’ adherence to malaria test results, shortens patients’ waiting time and leads to overall patient satisfaction. There is a need to establish RDT corners at OPDs of health facilities to implement pre-consultation testing. Plasmodium that are transmitted to people through the bites of infected female Anopheles mosquitoes. There are five parasite species that cause malaria in humans, however, Plasmodium falciparum and Plasmodium vivax pose the greatest threat, with Plasmodium falciparum being the most prevalent and the cause of most malaria-related deaths on the African continent [P. falciparum (80–90%), Plasmodium malariae (20–36%) and Plasmodium ovale (0.15%) [Malaria is a deadly disease caused by protozoan parasites of the genus ontinent . In Ghanmalariae 0–36% andAccording to Ezeonwu et al.  preventaMalaria is endemic in all regions of Ghana with a reported prevalence of 28% among children aged 6–59 months, based on microscopy , and treIn Ghana, malaria diagnosis is progressively being shifted from clinical to parasitological confirmation as the basis of treatment, and this is in compliance with global initiatives and recommendations such as the test, treat and track (T3) to scale up parasite-based diagnosis in all age groups . The iniTesting of patients immediately after triaging so that the patient sees the clinician with test results (pre-consultation testing) is likely to promote clinicians’ adherence to test results and also potentially shorten the waiting time at the health facility. The study therefore sought to assess whether implementation of pre-consultation testing would influence clinicians to adhere more to malaria test results, consider alternative diagnoses in the event of negative test results and lead to a reduction in time spent at the health facility by mothers/caregivers and their children age under 5 years in the Northern Zone of Volta Region, Ghana.The study was carried out at two government-owned district hospitals in two districts that were randomly selected in the Northern Zone of Volta Region of Ghana from November 2018 to January 2019. The Volta region stretches across all the ecological zones of the country: Coastal Savannah, Middle forest and Northern/Sahel Savannah. The regional prevalence of malaria among children under five years of age decreased from 25.2% (2014) to 24.5% (2018) [The region has 326 health facilities, of which 242 are public sector owned, 18 mission owned, 1 quasi-government , and 65 privately owned , 17. TheThe government hospitals in the Northern Zone of Volta Region were paired according to certain characteristics, including size, caseload, staffing, and availability of testing facilities. The names of each pair of hospitals were written on pieces of paper, wrapped into small balls and placed in a container. An independent person not participating in the study was asked to pick one wrapped paper from the container. The names of the randomly selected hospital pair were subsequently written on two separate pieces of paper and an independent person again asked to pick one. The hospital that was picked became the intervention hospital while its counterpart served as the comparator hospital.The study population comprised children aged 6 to 59 months with complaint of fever or history of fever reporting to the two government-owned hospitals as well as clinicians providing health care in those hospitals.A quasi-experimental study design was used for the study, which involved two randomly selected government-owned district hospitals, one of which was the setting for the intervention arm and the other, the setting for the comparator arm from November 2018 to January 2019. Trained nurses and data collectors assisted in data collection. The informed consent forms were translated into two local languages and administered to participants based on the language they were most comfortable with.For the intervention arm, an RDT corner was set up at an OPD to test all children aged 6 to 59 months complaining of fever or a history of fever before consultation with the clinician so that the clinician could see the patient with their test results. In the comparator arm, standard services that involve patients seeing a clinician before he/she decides whether to send the patient for testing or not, were maintained. In the laboratory in that setting when blood samples were being taken for microscopy, RDT was done concurrently. In the comparator arm, if after consultation with the patient, the clinician does not request for malaria testing but diagnoses an eligible child based on clinical symptoms, such child was excluded from the study.Data collection methods employed were quantitative. Questionnaires were administered to caregivers of children and clinicians providing care to the children under 5 years old. Clinicians were interviewed after the estimated sample size of mothers/caregivers was recruited, to avoid them being aware that the test results presented during the consultation was to determine their level of adherence to the test results. This was done to avoid bias.The dependent variables were clinicians’ adherence to test results and time spent in the health facility by the children. The independent variables considered in this study comprised socio-demographic factors  among the children, age of parent/guardian of child, geographic location, education level, and occupation) and other epidemiologic factors (fever or history of fever (within 48 h).Based on the prevalence of malaria among children under 5 years of age by RDT in the Volta region [Trained nurses and laboratory technicians in the study arms tested children aged 6 to 59 months for malaria using the RDTs. In the intervention arm, trained nurses tested for malaria using RDT before consultation with the clinician, while in the comparator arm, the children were tested in the laboratory after consultation with the clinician.P. falciparum in the finger prick blood sample in the diagnosis of malaria infection. Each child was given a specific identification number (ID). The unique ID was written on the cassette before beginning the test on each eligible child. If the child was right-handed, the 4th finger on the left hand was chosen for finger pricking. Likewise, the right 4th finger was chosen if the child was left-handed. The result was read within 20 min and recorded on the case record form.The SD BIOLINE Malaria Ag P.F (HRP2/pLDH) test kits were used for the rapid qualitative detection of histidine-rich protein II (HRP-II) antigen and lactose dehydrogenase (pLDH) from malaria In both settings, a research blood slide was taken from each participant at the time of testing for expert microscopy. Thick blood films were prepared on each glass slide using 10 µL of blood, evenly spread to cover an area of 15 × 15 mm of the slide. The smear was stained with 1% Giemsa for 25 to 30 min and then examined under oil immersion with a light microscope (magnification 100×). The slides were double-read by trained microscopists. Parasite species were estimated by counting the number of parasites per 200 white blood cells (WBCs) in the thick film. Counts of gametocyte were taken against 500 WBCs in determining gametocyte density per microlitre blood .A sample was considered negative only after 200 high power fields had been read. Parasite counts were converted to parasites per µL, with the assumption that there was an average of 8000 leukocytes per µL of blood. In cases where there was more than 50% discrepancy between parasite counts from the two microscopists readings or when there was a discrepancy qualitatively (positive or negative), a third microscopist read the slide and his/her reading was final and used in the analysis of parasite density. As part of quality control monitoring, randomly selected stained slides from each batch of slides were given to an independent expert microscopist to read. The slides were read on the same day after a day’s field work. Two nurses from hospital were attached to the project. When children were found to have malaria parasitaemia after reading the research slides, they were notified for follow-up purposes.Data from participants were recorded on specified data collection tools, and these were verified for consistency, completeness and accuracy at the study site. All data collected were entered into EpiData 3.1 software, cleaned and exported to STATA version 14.1 for analysis. Simple frequency and percentages were used for categorical variables. Statistical significance was considered based on a precision of 5% and a confidence interval of 95%. The results were displayed in tables and graphs. IDs were assigned to participants for data entry into the database. This was to ensure confidentiality since their names were not captured into the database. The PI was the only one who had access to the entered database, which was encrypted with a special password. This was to ensure that data are secured and properly managed.Eight-hundred and seventy-eight (878) children aged 6 to 59 months with complaint of fever or history of fever were recruited for the study with 439 children each from the intervention and comparator arm. The overall mean age of the children was 3.0 ± 1.4 months. Of the 878 children, 495 (56.4%) were males. More than half of the children 680 (77.5%) had normal body temperature (< 37.5 °C) at the time of reporting to health facilities. Most of the caregivers 763 (86.9%) owned a LLIN and 526 (68.9%) of children had slept inside an LLIN the night before the survey.The overall mean age of the mothers/caregivers was 31.6 ± 8.9 years. There were more mothers/caregivers with junior high school education in the intervention arm than the comparator arm (47.6 vs. 40.3%). More than half of the mothers/caregivers (65.4%) were married in both study arms. Most mothers/caregivers (81.4%) were Christians. More than half of the mothers/caregivers (57.3%) were of Ewe ethnic extraction and 43.5% were traders.The overall mean age for clinicians was 28.9 ± 5.5 years. More than half of the clinicians (66.7%) were under 30 years of age. Most of the clinicians (91.7%) were males. All clinicians (100%) were first-degree holders and majority (83.3%) were Physician Assistants. Most (83.3%) were single and more than half (66.7%) had less than 2 years of work experience.Of the 878 children recruited, 280 31.9%) and 234 (26.7%) tested positive for malaria RDT and expert microscopy, respectively. Clinicians in the comparator arm did not send an approximate of 15% of the total eligible children for malaria testing, hence those children were excluded from the study. Overall, the observed prevalence of malaria in the children as indicated by the RDT was significantly higher than that of the microscopy    were still diagnosed with malaria as compared to 25 (8.4%) out of a total of 299 children who tested negative in the intervention arm   in both study arms Fig. . HoweverP. falciparum in the finger-prick blood sample in the diagnosis of malaria infection. Since the RDT picked up HRP2 antigens, this may have accounted for the increased prevalence observed using RDTs.The findings from this study showed that the overall prevalence of malaria among children aged 6 to 59 months with complaints of fever or history of fever based on RDT testing was higher than the prevalence based on microscopy (31.9 vs. 26.7%). The SD BIOLINE Malaria Ag P.F (HRP2/pLDH) test kits were used for the rapid qualitative detection of HRP-II antigen and pLDH from The difference in prevalence using different testing methods observed in the current study is similar to that of Lekweiry and others in their study carried out in Nouakchott, Mauritania where a malaria prevalence of 34.9 and 28.9% was found among children aged 6 to 59 months using RDT and microscopy testing, respectively .In Madagascar, Ratsimbasoa et al.  also fouThe prevalence of malaria as indicated by RDT was higher than microscopy in this current study as was found in the other studies. The difference in malaria prevalence found using the two testing methods (RDT and microscopy) could be due to persistence of circulating HRP2 antigens after malaria treatment, leading to treated patients still being identified as positive cases by RDT .Clinicians’ adherence in the intervention arm was significantly higher than that of those in the comparator arm . This finding agrees with that of Asiimwe et al.  in UgandThe current study found that in the intervention arm, 8.4% of the children aged 6 to 59 months reporting with fever or a history of fever were still diagnosed with malaria following a negative RDT result, compared to 21.2% in the comparator arm following microscopy test results. The findings differ from what was found by Manyando et al. . In GhanThe reason for the lower proportion of children under 5 years diagnosed with malaria following a negative RDT in the intervention arm in this current study compared to earlier studies could be that over time, clinicians may have begun to have more confidence in RDT results than they used to have since the other studies were carried out much earlier. However, the reason why clinicians still diagnosed children with malaria following negative RDT results could be partly attributed to lack of capacity by clinicians to adequately investigate other causes of fever , 25.With regard to the average time spent at the OPD, mothers/caregivers’ children in the intervention arm spent less time compared to those reporting at the comparator arm (2.6 vs. 3.4 h). The time spent by patients reporting to the intervention health facility is similar to that spent by patients in an earlier study conducted by Ogunfowokan and Mora  in Nigerp < 0.001) [In this study, the proportion of mothers/caregivers who viewed the waiting time at the OPD as “too much” was significantly lower in the intervention arm than in the comparator arm . The current study confirms the dissatisfaction of mothers/caregivers of children aged 6 to 59 months with the long waiting time in the health facility as a result of seeing the clinician before requesting for the test, as practiced in the comparator arm . In a si< 0.001) .Clinicians were interviewed after the estimated sample size of the mothers/caregivers were recruited, to avoid them being aware that the test results the children presented during the consultation were to determine their level of adherence to the test results. This was done to avoid bias.The medical superintendents in charge of the hospitals, were however made aware of this when permission was being sought and were assured of anonymity and confidentially of the study. It may however, be likely, though remotely so, that they might have informed some clinicians of the aim of the study.Additionally, baseline data on patients waiting time were not collected in both hospitals prior to the introduction of pre-consultation testing in the intervention arm. Even though the hospitals were paired according to certain characteristics such as size, case load, staffing, and availability of testing facilities, there was a possibility of other differences in the two hospitals which might have contributed to the longer consultation time in the comparator arm.Pre-consultation testing for malaria at an OPD significantly improved clinicians’ adherence to malaria test results, shortened patient waiting time and led to an overall patient satisfaction compared to post-consultation testing in the Northern Volta region of Ghana. Pre-consultation testing resulted in a significant reduction in clinicians’ diagnoses of malaria following negative test results. Clinicians and caregivers of children aged 6 to 59 months in both settings indicated a preference to have children tested at OPD of the hospitals before consulting with a clinician. Inclusion of RDT for children aged 6 to 59 months with fever at the triaging area of OPDs will lead to an improvement in the quality of diagnosis and treatment of malaria.


Plasmodium falciparum blood-stage antigens, MSP-119, MSP-2, MSP-3 and AMA-1. We used the assay to study the kinetics of the MBC response after an acute episode of malaria and up to one year following treatment in travelers returning to Sweden from sub-Saharan Africa. We show that the FluoroSpot assay can detect MBCs to all four merozoite antigens in the same well, and that the breadth and kinetics varied between individuals. We further found that individuals experiencing a primary infection could mount and maintain parasite-specific MBCs to a similar extent as previously exposed adults, already after a single infection. We conclude that the multiplexed B-cell FluoroSpot is a powerful tool for assessing antigen-specific MBC responses to several antigens simultaneously, and that the kinetics of MBC responses against merozoite surface antigens differ over the course of one year. These findings contribute to the understanding of acquisition and maintenance of immune responses to malaria.Memory B cells (MBCs) are believed to be important for the maintenance of immunity to malaria, and these cells need to be explored in the context of different parasite antigens and their breadth and kinetics after natural infections. However, frequencies of antigen-specific MBCs are low in peripheral blood, limiting the number of antigens that can be studied, especially when small blood volumes are available. Here, we developed a multiplexed reversed B-cell FluoroSpot assay capable of simultaneously detecting MBCs specific for the four  Acquisition of clinical immunity to malaria is achieved after multiple infections in individuals living in areas with high transmission , 2. The P. falciparum as well as the kinetics and breadth of MBC responses after a natural infection is yet to be elucidated. Few studies have investigated MBCs longitudinally within an individual after a malaria episode  are considered long-lived and can be maintained for decades in the absence of circulating antibodies . At a he episode , 15. Nonexposure . In ordeP. falciparum antigens as well as limited sample volumes, especially in children, novel highly sensitive tools are required for analysis. Recently, we described a reversed B-cell FluoroSpot assay for simultaneous detection of human antigen-specific MBCs against three different pathogens . Patients who suffered from treatment failure and presented with recrudescent malaria during treatment, were successfully treated with a second course of either artemether–lumefantrine (Riamet®) or mefloquine . The primary infected individuals were all Swedish natives with no previous malaria diagnosis, and no or only short-term cumulative residency in malaria endemic areas (range 0-2 years). The previously exposed individuals were Swedish residents born in Sub-Saharan Africa who previously lived in a malaria-endemic area for an extended period of time (range 18-39 years) and who reported that they had multiple malaria infections before moving to Sweden. Also, five healthy adults who had never visited malaria endemic areas were included as unexposed controls. In addition, 1-month follow-up samples from five adults of African origin within the cohort with high plasma levels of antibodies against the four selected antigens  was collected in sodium-heparin or EDTA tubes  at each visit. Peripheral blood mononuclear cells (PBMC) were isolated from 35 mL whole blood using Ficoll density centrifugation as previously described . Five miP. falciparum merozoite antigens were expressed recombinantly in a mammalian expression system as previously described , P50498; MSP-2 (FC27-type), P19599; MSP-3, Q8IJ55 and AMA-1, Q7KQK5. For MSP-2, the 3D7 and FC27 type antigens were expressed and tagged with the same peptide tag and referred to as MSP-2 when used in combination at equal amounts. The antigens were tagged with the following peptide tags described previously , with GAL; MSP-3 with WASP and AMA-1 with TWIN-Strep-tag®  (amino acid sequence WSHPQFEKGGGSGGGSGGSAWSHPQFEK). In potential N-linked glycosylation sites, (NXS/T), serine (S) or tyrosine (T) were replaced by alanine (A) to avoid epitope interference. The mouse IgG kappa leader sequence (METDTLLLWVLLLWVPGSTGD) was added to each sequence to ensure protein secretion. Also, tag control antigens; cow IFN-γ tagged with peptide tag BAM, woodchuck IFN-γ tagged with GAL, dog IFN-γ tagged with WASP, and horse IFN-γ tagged with TWIN-Strep-tag® were expressed separately as described  at the N-terminus to be used for ELISA. Synthesis and cloning of sequences into plasmid pcDNA3.1/Zeo(−)  was performed by GeneScript . Transient transfection was performed using the Expi293 transfection system (Thermo Fisher Scientific) as previously described  (STXT-550), all diluted to 0.5 µg/mL in PBS/BSA supplemented with filtered human plasma diluted 1:100, were added to the wells and incubated for 1 hr at RT. Human plasma was added to block coating antibody from potentially binding anti-tag detection mAb. The frequency of total IgG-producing cells was analysed using 25,000 pre-stimulated PBMCs in two separate duplicate wells. To detect IgG-producing cells, 100 μL/well of biotinylated mAb anti-human IgG (MT78/145-biotin) diluted to 0.5 μg/mL, were added followed by Streptavidin-550 in PBS/BSA. After a final wash, 50 μL of Fluorescent enhancer (Mabtech AB) was added to plates and incubated for 10 minutes at RT before the plates’ underdrain were removed and plates were left to dry in the dark at RT. The FluoroSpot plates were analysed using the Mabtech IRIS™ reader system equipped with Apex™ software version 1.1.7. The relative spot volume (RSV) of each counted spot was determined by the Apex™ and presented as arbitrary units. Definition of assay disqualification of a sample was set to <1000 total IgG spot forming units (SFU) per 250,000 cells. The threshold for MBC positivity was defined as the mean reactivity of healthy unexposed controls plus 2 standard deviations (SD). The percentage of antigen-specific MBCs was calculated by dividing the number of antigen-specific spots with the number of total IgG spots.A schematic presentation of the FluroSpot assay is shown in 19, MSP-2 (3D7), MSP-2 (FC27), MSP-3, AMA-1, as well as a tagged control antigen (horse IFN-γ), were analysed by ELISA. The antigens were expressed with the same amino acid sequence as the antigens used for FluoroSpot with the difference that they were all tagged with a TWIN-Strep® tag. High-binding polystyrene 96-well ELISA plates were coated with 100 μL/well of Strep-Tactin®XT (IBA BioScience) diluted to 1 μg/mL in PBS and incubated overnight. The following day, plates were blocked with 100 µL incubation buffer consisting of PBS supplemented with 0.1% BSA and 0.1% Tween 20 , for 1 hr. Next, the plates were washed using an automated ELISA washer, followed by the addition of 100 μL TWIN-Strep® tagged variants of the above-mentioned antigens diluted 1:5 in incubation buffer. Next, the plates were washed using an automated ELISA washer, followed by the incubation of 100 µL plasma diluted to 1:2000 in incubation buffer for 1hr at RT. Following wash, 100 µL of horseradish peroxidase conjugated mouse anti-human IgG mAb (Mabtech AB) diluted to 0.5 µg/mL in incubation buffer was added to the plates and incubated for 1 hr and then washed as above. Development of signal was done by adding 100 µL 3,3’,5,5’-Tetramethylbenzidine (TMB) substrate solution (Mabtech AB) to the plates for 10 min, after which the reaction was stopped by addition of 100 µL ELISA Stop solution (Mabtech AB). Plates were read in an Emax ELISA reader at 450 nm and analysed using ELISA software SoftMax pro . The threshold for seropositivity was defined as the mean reactivity of five healthy malaria unexposed controls plus 2 SD.Antibody levels, in plasma samples collected in parallel with the PBMCs at each time point, against antigens MSP-1E.coli-derived antigens described elsewhere , MSP-2_Dd2 , MSP-3_3D7 or AMA-1_3D7 at 1 μg/mL in PBS and incubated overnight. After blocking and washing, a pool of plasma from individuals considered semi-immune against P. falciparum malaria was added in serial dilutions. The plates were developed and analysed as described for the peptide tag-based ELISA. A schematic presentation of the two ELISA formats is shown in In order to compare the antigenicity of the expressed antigens with peptide tags and purified antigens, an indirect ELISA was performed using the purified lsewhere . In brieE. coli-derived purified antigens, associations between %MBC/total IgG and SFU (goodness of fit was shown as r), and variability between replicate wells. A mixed-effects linear regression model adjusted for categorical time point of sample together with subject specific random intercept and time slope was used to compare differences between groups regarding %MBC/total IgG, RSV, antibody levels, SFU total IgG, and breadth, but also if MBC responses in patients suffering from treatment failure differed from other patients in that group. A p value <0.05 was considered as significant.Statistical analyses were performed using GraphPad Prism version 8.3  and STATA MP version 16.0. The Mann-Whitney U-test was used to compare reproducibility between tests, duplicates and singleplex vs multiplex regarding SFU and RSV as well as differences in %MBC/total IgG or antibody levels between categorical timepoints. Spearman correlation was used to analyse associations between logarithmically transformed %MBC/total IgG, RSV or antibody levels throughout the study period, associations between immunogenicity of newly expressed antigens and P. falciparum merozoite antigens were expressed recombinantly in a mammalian expression system with either a peptide or TWIN-Strep® tag. Antigen expression was verified by Western Blot, with bands detected at expected molecular weights  and two patients had severe malaria .When time since residency in an endemic area was correlated to MBC responses for previously exposed individuals, no significant association were detected against any antigen with the exception of MSP-1 that showed a positive correlation with time since residency in an endemic area   , MSP-2 (FC27), MSP-3 and AMA-1 were measured by peptide tag ELISA with TWIN-Strep® tagged variants of the antigens (Antibody responses in plasma against MSP-1antigens . When anLimited correlation was observed between plasma antibody levels measured by ELISA and MBC responses measured by FluoroSpot (19 with positivity in 18 (90%) individuals, followed by AMA-1 and MSP-2 in 17 (85%), and MSP-3 in 14 (70%) of the individuals , respectively. The number of individuals that were considered MBC positive against an antigen, on at least one time point during the study period, was highest for MSP-1ividuals Figure 9The breadth of MBC responses was lowest at the acute time point, with responses to a median of 1 (range 0–3) antigen, and then increased at the later time points. The breadth of MBC responses was highest at the 1 month-follow up (median 3 range 0–4) and tended to decline thereafter Figure 9The breadth of antibody responses in plasma was highest at the 10-day and 1-month follow-up but then declined at the later timepoints Figure 919, MSP-2, MSP-3, and AMA-1) in PBMCs from individuals followed prospectively after an acute episode of P. falciparum malaria. We demonstrated the quality and functionality of the multiplex assay to study antigen-specific MBC responses ex vivo. By analysing the MBC response at several time points over one year following treatment in a malaria free setting, we could study the kinetics of the response during and after the acute infection without the risk of re-infection. We show heterogeneity in responses to the four included antigens within and between individuals, and that both primary infected and previously exposed individuals were able to acquire and maintain merozoite antigen-specific MBCs one year after natural acute infection.Here, we developed a reversed B-cell FluoroSpot assay that simultaneously detects antigen-specific MBCs to the four different merozoite antigens , as determined by ELISpot several years after infection  of individuals had MBC responses against all four merozoite antigens at a single time point. Heterogeneity of the MBC responses, also within individuals, may be explained by low frequencies of MBCs in circulation, falling under the detection limit of the assay in individual samples. Importantly, most individuals (65%) were positive against all antigens at some time during the one year follow up, demonstrating the detection sensitivity of our assay as well as the advantage of following patients over time compared to investigating just a single time point.All, but one individual (a primary infected traveller) followed after acute 19 and AMA-1 that were maintained for an extended period of time  observed after a primary infection was remarkably high in some individuals. A potential reason for this is that MSP-1 is the most abundantly expressed protein on the parasite surface , project grants from the Swedish Research Council (VR MH 201502977 and 201802688) and the Stockholm County Council (ALF 20150135 and 20180409).PJ, CSm, and NA are employed by Mabtech AB, Sweden. Several of the reagents and the FluoroSpot reader system used in this study are produced by Mabtech. Mabtech has no influence on the content of this study.The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Malaria in pregnancy increases the risk of deleterious maternal and birth outcomes. The use of ≥ 3 doses of sulfadoxine-pyrimethamine (SP) for intermittent preventive treatment of malaria (IPTp-SP) is recommended for preventing the consequences of malaria during pregnancy. This study assessed the effect of IPTp-SP for prevention of malaria during pregnancy in low transmission settings.A cross-sectional study that involved consecutively selected 1161 pregnant women was conducted at Mwananyamala regional referral hospital in Dar es Salaam. Assessment of the uptake of IPTp-SP was done by extracting information from antenatal clinic cards. Maternal venous blood, cord blood, placental blood and placental biopsy were collected for assessment of anaemia and malaria. High performance liquid chromatography with ultraviolet detection (HPLC-UV) was used to detect and quantify sulfadoxine (SDX). Dried blood spots (DBS) of placental blood were collected for determination of sub-microscopic malaria using polymerase chain reaction (PCR).In total, 397 (34.2%) pregnant women reported to have used sub-optimal doses (≤ 2) while 764 (65.8%) used optimal doses (≥ 3) of IPTp-SP at the time of delivery. The prevalence of placental malaria as determined by histology was 3.6%. Submicroscopic placental malaria was detected in 1.4% of the study participants. Women with peripheral malaria had six times risk of maternal anaemia than those who were malaria negative . The geometric mean plasma SDX concentration was 10.76 ± 2.51 μg/mL. Sub-optimal IPTp-SP dose was not associated with placental malaria, premature delivery and fetal anaemia. The use of ≤ 2 doses of IPTp-SP increased the risk of maternal anaemia by 1.36-fold compared to ≥ 3 doses .The use of < 2 doses of IPTp-SP increased the risk of maternal anaemia. However, sub-optimal doses (≤ 2 doses) were not associated with increased the risk of malaria parasitaemia, fetal anaemia and preterm delivery among pregnant women in low malaria transmission setting. The use of optimal doses (≥ 3 doses) of IPTp-SP and complementary interventions should continue even in areas with low malaria transmission. Despite the efforts to control and eliminate malaria worldwide, malaria in pregnancy (MiP) has remained a significant contributor of maternal, neonate and infant morbidity and mortality. The recent World Health Organization (WHO) malaria report indicated that 219 million malaria cases and 435,000 deaths were reported worldwide, of which 80% were from sub-Saharan Africa and India [Sulfadoxine (1500 mg)/Pyrimethamine 75 mg) is given to pregnant women as a single therapeutic dose on direct observed therapy (DOT) during visits to the antenatal clinics (ANC). The drug is readily absorbed (bioavailability > 90%) and reaches peak plasma concentration of about 183 μg/mL (sulfadoxine) and 0.55 ng/mL (pyrimethamine) 2–8 h after oral administration . Sulfado5 mg is gdihydrofolate reductase (Pfdhfr) and dihydropteroate synthase (Pfdhps) genes, rendered the previous recommended 2-doses ineffective in averting adverse birth and maternal outcomes due to MiP [The beneficial effect of IPTp-SP is believed to be due to suppression rather than complete clearance of parasites in the peripheral and placenta , 11. Thee to MiP –19. Inste to MiP –22. HoweThe impact of IPTp-SP on birth outcomes among pregnant women living in moderate to high malaria transmission areas have been widely studied. There still exists the question on the benefit of using IPTp-SP in areas with substantial reduction of malaria transmission. To cover this knowledge gap, the study describes the influence of optimal and sub-optimal uptake of IPTp-SP doses on MiP as well as on the adverse birth outcomes among pregnant women living in low malaria transmission in Dar es Salaam, Tanzania.This was a cross-sectional study conducted between April and November, 2018 and involved 1161 pregnant women with at least 18 years of age, admitted in the delivery unit at Mwananyamala regional referral hospital in Kinondoni Municipality, in Tanzania. Pregnant women in delivery wards were recruited consecutively. Those who had complicated pregnancy with high risk of haemorrhage, preeclampsia, eclampsia, HIV positive, with incomplete ANC cards  was used to collect information on socio-demographic characteristics such as place of residence, age, gender, marital status and education level; obstetric characteristics; the use of anaemia preventive measures example FEFO  and mebendazole, 500 mg; malaria preventive measures including IPTp-SP, mosquito repellents or spray and ITN; the number of IPTp-SP doses used as documented on ANC card; history on the previous malaria infection and anti-malarial used; and, birth outcomes. Gestation age was determined by last normal menstrual period and documented.® Hb 201 + HemoCue AB, Angelholm, Sweden. The WHO cut off points on haemoglobin were used to characterize maternal and fetal haemoglobin status as normal or anaemia [Three EDTA tubes were used to collect maternal blood, cord blood  and placental blood. The EDTA tubes were inverted ten times (to enable mixing of whole blood with EDTA) then transferred to the laboratory at Mwananyamala Regional Referral Hospital for malaria and haemoglobin examination. Maternal and fetal Hb levels were determined using HemoCue anaemia . Pregnan anaemia , 22.® Hb 201 + HemoCue AB, Angelholm, Sweden. About 5 μL of blood was used for testing malaria infection using malaria Rapid diagnostic test, (RDT), SD BIOLINE Malaria Ag P.f/pan, Standard Diagnostics, INC. Microscopy was used to confirm the results of RDT. Thick and thin blood smears were stained with 2% Giemsa. Microscopic examination was performed by two competent laboratory technologists, any discordant in results was resolved by the third reader. A blood smear was considered negative when the examination of 100 high power fields did not reveal asexual parasites.Laboratory analysis of blood samples Maternal and fetal Hb levels were determined using HemoCue3 was placed in a container with 10% neutral buffered formalin and stored at room temperature for transfer and processing at Muhimbili University of Health and Allied Sciences (MUHAS) pathology laboratory. Paraffin-embedded placental specimens were sectioned, stained with haematoxylin and eosin (H&E) and Giemsa. Malaria parasites and pigments were examined under light microscopy. Malaria parasites were identified by their presence in the erythrocytes in intervillous space. On the other hand, malaria pigments were identified by their presence in the erythrocytes and monocytes in intervillous space and pigment in fibrin [After delivery, placenta was sliced using a disposable lancet halfway between the edge of placenta and insertion of the cord. The placental biopsy with approximately 2 cmn fibrin . Two exp®3MM, Maidstone, UK), air dried overnight and preserved in plastic bags for PCR analysis. After histological analysis of placental biopsy for placental malaria, 286 DBS samples with negative placental malaria were randomly selected to include participants who used ≤ 2 and ≥ 3 IPTp-SP doses. The 286 DBS samples were used to determine submicroscopic malaria infection by PCR. The DBS were transported to the laboratory at the National Institute for Medical Research, Tanga Centre for detection of submicroscopic malaria infection. The DNA was extracted from DBS using the QIAamp DNA Minikit  following the manufacturer’s protocol. DNA was eluted in 150 μl of buffer and samples were stored at − 20 °C freezer until time of use. The Plasmodium species were identified using nested PCR according to the Snounou et al. protocol [Two drops of placental blood were spotted on a filter paper . Spearman’s correlation was used to establish the relationship between continuous variables such as maternal hemoglobin concentration and SDX concentration. Variables that had p-value ≤ 0.2 were subjected to multivariate analysis to establish their influence on the relationship between IPTp-SP doses and outcomes of interest. Multivariable logistic regression was performed to determine the effect of IPTp-SP doses on placental malaria, maternal anaemia, fetal anaemia and preterm delivery. Significance level was set at 0.05 and the confidence level at 95%. Exposure variables that had p-value < 0.05 were considered significant predictor of the outcome variables. The IPTp-SP doses were grouped as sub-optimal and optimal doses when participants used ≤ 2 doses and ≥ 3 doses, respectively. Analysis was conducted using a Statistical Package for Social Sciences (SPSS) program version 23.0.The total of 1161 pregnant women participated in the study with median (IQR) age of 25 (18–44) years old. Most of them were married (73.7%) with primary education level (60.9%) and attended ANC visits at least 4 times (66.9%) during their recent pregnancies. They reported to have used various malaria preventive measures, such as mosquito sprays/repellents (57.9%) and ITN (98.1%), and anaemia preventive strategies including mebendazole (96.6%) and FEFO (97.9%)  gestation age of 38.8 ± 1.6) weeks with birth weight of 3.1 (± 0.46) Kg. The proportions of primigravida, secundigravida and multigravida among the study participants were 38.4%, 28.3% and 33.3%, respectively  women used ≤ 2 doses while 764 (65.8%) used ≥ 3 doses of IPTp-SP Fig. . Most of% women uPlacental malaria by histology was detected in 42 3.62%) out of the 1161 study participants. Three (7.1%) out of the 42 pregnant women with peripheral malaria had placental malaria (p = 0.002), accounting for 21-fold risk of placental malaria than those without peripheral parasitaemia . The prevalence of placenta malaria with active infection, active-chronic infection and past infection were 2.5%, 0.4% and 0.5% respectively. Out of 397 pregnant women who used ≤ 2 doses of IPTp-SP, 16 (4.0%) of them had placental malaria. For 764 pregnant women who used ≥ 3 doses of IPTp-SP, 26 (3.4%) of them had placental malaria. Sub-optimal doses (≤ 2 doses) of IPTp-SP did not increase the risk of placental malaria among pregnant women (p = 0.97)  had submicroscopic placental malaria. Of these participants with submicroscopic placental malaria, 3 (1.58%) of them used ≥ 3 IPTp-SP doses while 1 (1.04%) used ≤ 2 IPTp-SP doses. PCR positive samples revealed that, all the submicroscopic infections were due to Out of 1161 pregnant women, the prevalence of maternal anaemia and fetal anaemia was 43.8% and 10.1%, respectively. Peripheral malaria was significantly associated with maternal anaemia and not fetal anaemia. Women with peripheral malaria had six times risk of maternal anaemia than those who had no malaria infection . In multivariable analysis, the use of ≤ 2 IPTp-SP doses increased the risk of maternal anaemia by 1.36 times higher compared to the use of ≥ 3doses , . Further analysis revealed that, pregnant women who had anaemia were 2 times at increased risk of delivering anaemic babies . Other characteristics of pregnant women such as age, marital status, education level, number of ANC visits, use of FEFO, mebendazole, ITN, mosquito spray/repellents, gestation age, gravidity and placental malaria were not associated with maternal and fetal anaemia.Also, the risk of preterm delivery was not increased by the use of sub-optimal IPTp-SP doses, however, factors such as primigravidity and mosquito spray/repellents were associated with preterm delivery as reported previously . Women wSDX was detected in 218 (92.0%) out of 237 participants. A majority (60.1%) of those who had detectable SDX used ≥ 3 doses of IPTp-SP. In the analysed samples (n = 237), 79 (33.3%) samples had detectable SDX levels but could not be quantified (plasma concentration was < 3 μg/ml). Three (1.3%) participants had detectable and quantified SDX concentration despite of reporting to have not taken any dose of IPTp-SP throughout their recent pregnancies. Out of 19 (8.0%) women who had undetectable SDX in plasma, 13 (68.4%) used ≤ 2 doses of IPTp-SP. Six (2.5%) participants who reported to have used ≥ 3 doses of IPTp-SP had no detectable SDX in plasma. The overall geometric mean plasma SDX concentration was 10.76 ± 2.51 μg/mL. The geometric mean concentration for women who used ≥ 3 IPTp-SP doses was 10.46 ± 2.50 μg/mL, while those who used ≤ 2 doses was 11.24 ± 2.54 μg/mL. There was no difference in the geometric mean of plasma concentration between women who used sub-optimal versus optimal IPTp-SP doses (p = 0.65) . In addition, the differences in SDX plasma concentration at birth had no effect on placental malaria among the study participants (p = 0.24) Table .Table 5EThere was a slight increase in maternal Hb concentration with plasma concentration of SDX at delivery, but this relationship was weak (spearman’s correlation = 0.02) Fig. .Fig. 2PlThis study highlights the effect of sub-optimal IPTp-SP doses on placental malaria, maternal anaemia and fetal anaemia in malaria hypoendemic region (1.1%). The effect of plasma SP concentration in relation to maternal and birth outcomes was also determined. The prevalence of placental malaria, maternal anemia, fetal anemia and preterm delivery was 3.6%, 43.8%, 10.1% and 7.8% respectively. The use of sub-optimal IPTp-SP doses increased the risk of maternal anaemia by 1.36 times higher than optimal IPTp-SP doses.Majority of pregnant women reported to have used the recommended optimal doses (≥ 3 doses) of IPTp-SP during pregnancy. The improved uptake of IPTp-SP ≥ 3 could be attributed to the increase in sensitization of SP use for pregnant woman attending the ANC. Previous report on the use of IPTp-SP in Tanzania indicated the low prevalence of 26% for the use of ≥ 3 doses of IPTp-SP which is below 60% target by the ministry responsible for health .This study was conducted in an urban area where the use of mass media in advertising the importance of IPTp-SP use is highly publicized. Moreover, the availability of well-trained health care providers and healthcare facilities being close to households as well as equipped health facilities might have contributed to improved uptake of IPTp-SP in pregnant women. The use of ≥ 3doses of IPTp-SP was significantly associated with ANC attendance. Similar, findings have been reported by other studies indicating that frequent contact with health care providers increased the chances of more intake of SP doses among pregnant women .In this study, there was lower prevalence of placental malaria 3.6%). This is almost half (6.6%) of the prevalence of placental malaria that was reported in the same catchment area in 2010/2012 . The red.6%. ThisThe current study observed an increased risk of placental malaria among pregnant women with peripheral malaria. The risk of placental malaria among pregnant women with peripheral malaria has been well studied , 31–33. SDX was used as a proxy of uptake of SP owing to its long half-life of about 10 days , 8. SDX P. falciparum [The findings of this study, indicate that, the use of sub-optimal doses of IPTp-SP increased the risk of maternal anaemia in low transmission settings. The association of sub-optimal doses of SP with anaemia may result because these women received fewer FEFO doses or if they attended fewer ANC visits. Also, if a women delivered preterm she may not have been able to receive a third/fourth IPTp-SP dose. As Hb is often lower in mid-pregnancy due to plasma volume dilution, this might also relate to the observation of lower Hb in women received ≤ 2 doses. These results are in contrast to findings by Mosha and his colleagues who reported lack of effect of IPTp-SP doses on maternal anaemia . The diflciparum , 39. Sevlciparum , 41. Inflciparum .Individuals who are anaemic are more vulnerable to infections . In thisIn this study, pregnant women who were anaemic had increased risk of delivering anaemic babies . This association has also been reported in previous studies where the risk of fetal anaemia increased with severity of maternal anaemia , 44. FetPfdhps-A581G in combination with the quintuple mutant) [The use of sub-optimal or optimal doses of IPTp-SP was not associated with increased risk of fetal anaemia in this study. These findings are similar to those reported by Abrams et al. , indicat mutant) . About 9 mutant) . Despite mutant) . In the Since 90.7% of women who used at least one dose of IPTp-SP had detectable SDX at the time of delivery, this observation increased our confidence in using the documented number of doses on ANC cards and that majority of them took the last dose by DOT at least at the 28th week of gestation. Presence of SDX in plasma during pregnancy (from early in the 2nd trimester) is necessary to clear peripheral and placental malaria that subsequently reduces the risk of maternal anaemia and adverse birth outcomes , 49.This was a cross sectional study that involved large number of pregnant women at delivery. Adherence on the use of IPTp-SP could not be assessed as the number of doses reported was based on SP doses documented in the ANC cards. Despite this limitation, the detection of plasma SDX at delivery was useful in authenticating the information on the use of IPTp-SP among study participants. Due to few women with positive submicroscopic malaria parasites, the impact of IPTp-SP doses on submicroscopic placental malaria was not ascertained. Assessment of plasma SDX was done at delivery. Plasma SDX samples taken at different time intervals during pregnancy would have provided more reliable results to also assess adherence to IPTp-SP among pregnant women. However, based on the long half-life of SDX, the results of the present study are relevant for IPTp-SP doses that were taken from 20th to 40th week of gestation age.The use of sub-optimal doses of IPTp-SP increased the risk of maternal anaemia in malaria hypoendemic region in Tanzania. The use of sub-optimal IPTp-SP doses was not associated with increased risk of malaria parasitaemia, fetal anaemia and preterm delivery among pregnant women in low malaria transmission setting. Uptake of optimal doses (≥ 3 doses) of IPTp-SP and complementary interventions should continue while searching for alternative malaria preventive strategies to IPTp-SP in malaria hypoendemic regions. Early malaria case detection and prompt treatment with effective anti-malarial drugs is highly encouraged to prevent severe malaria and deleterious clinical outcomes in pregnant women.


Anopheles pseudopunctipennis in previously endemic areas in Chile require an active malaria surveillance programme.Chile is one of the South American countries certified as malaria-free since 1945. However, the recent increase of imported malaria cases and the presence of the vector pfk13, pfcrt and pfmdr1) and microsatellite (MS) analysis were performed in confirmed Plasmodium falciparum samples and results were related to origin of infection.Specimens from 268 suspected malaria cases—all imported—collected between 2015 and 2018 at the Public Health Institute of Chile (ISP), were diagnosed by microscopy and positive cases were included for epidemiological analysis. A photo-induced electron transfer fluorogenic primer real-time PCR (PET-PCR) was used to confirm the presence of malaria parasites in available blood samples. Sanger sequencing of drug resistance molecular markers  and three mutant pfmdr1 genotypes . MS analysis confirmed that all P. falciparum samples presented different haplotypes according to the suspected country of origin. Four patients with P. vivax infection returned to the health facilities due to relapses.Out of the 268 suspected cases, 65 were The timely detection of polymorphisms associated with drug resistance will contribute to understanding if current drug policies in the country are appropriate for treatment of imported malaria cases and provide information about the most frequent resistant genotypes entering Chile. Plasmodium. The four species of Plasmodium that commonly infect humans are Plasmodium falciparum, Plasmodium vivax, Plasmodium ovale, and Plasmodium malariae. The fifth human malaria species is Plasmodium knowlesi, known to cause simian malaria, which has been reported in nearly all countries from Southeast Asia and in travellers visiting these countries [Malaria is a mosquito-borne infectious disease caused by parasites of the genus ountries , 2.P. vivax and P. falciparum, with 773,000 malaria cases estimated in 2017; 97% of these cases were found in Brazil, Colombia, Peru, and Venezuela [In 2017, there were 219 million estimated cases of malaria worldwide, resulting in 435,000 deaths [enezuela , 7.Anopheles pseudopunctipennis) control and the prompt treatment of malaria patients [Chile has been certified as a malaria-free country by the World Health Organization (WHO), with no indigenously-acquired cases reported since 1945 . This wapatients . Betweenpatients . MalariaAn. pseudopunctipennis  breeding sites have been found in close proximity to residential areas in Valle de Azapa, Valle de Lluta, Quebrada de Camarones and Chaca in the Arica y Parinacota Region and in the Tarapacá Region. Additionally, González and Sallum, reported the presence of a new species of Anopheles with unknown vector potential in Atacama Region, northern Chile [Currently, rn Chile .P. falciparum infected patients in countries where CQ and SP resistance has been reported [P. vivax infections is chloroquine plus primaquine (CQ + PQ), with a variation of 7 or 14 days of therapy with PQ depending on the country [For many years, the recommended malaria treatment was chloroquine (CQ) followed by sulfadoxine–pyrimethamine (SP) until resistant parasites spread worldwide. Currently, artemisinin-based combination therapy (ACT) is the first-line treatment recommended by the WHO for reported . HoweverP. falciparum isolates harbouring the C580Y mutation in P. falciparum kelch 13 gene were reported in samples collected in 2010 in Guyana [Publications reporting delayed parasite clearance following ACT in Southeast Asia –13 placen Guyana . This mun Guyana , 15, 16.Plasmodium species, mixed infections, and to assess the drug resistance profile of parasites. Additionally, microsatellite (MS) markers were analysed in P. falciparum samples to explore the presence of clusters of cases and/or polyclonal infections.While malaria transmission is not known to occur in Chile, importation of malaria cases from other South American countries and other parts of the world requires proper monitoring in order to inform treatment policy. In this study, a molecular characterization of parasites from imported malaria cases was undertaken to confirm infecting As part of national malaria surveillance, samples from suspected malaria cases throughout Chile are collected from public and private health centers and sent to the Public Health Institute of Chile (ISP) for diagnosis. A database search was performed at ISP to collect epidemiological information of patients with suspected malaria from January 2015 to December 2018 using identifiers such as: age, gender, travel history, malaria treatment, health center and region of origin. This subsequent work was part of public health surveillance and therefore Institutional Review Board (IRB)/consent was not needed, and personal information was excluded for publication.Malaria infections in these samples at the time of diagnosis were determined by microscopy using Giemsa-stained blood smears performed at the ISP. All malaria positive patients were treated with atovaquone–proguanil and where possible, patients were followed up to ascertain clearance of their infection and were requested to report to the clinic if symptoms returned. Each health centre and treating physicians defended when and how to take additional samples for follow up, since this determination is not within the powers of the ISP.Plasmodium spp. were confirmed through microscopy using Giemsa-stained blood smears. For molecular analysis, 46 samples were included, which was the total number of available blood samples for subsequent analysis in the laboratory. These 46 samples corresponded to 39 different patients.This descriptive study used data from 268 suspected malaria cases received by the ISP during 2015–2018, period in which 65 positive patients with the CT value of < 40 was considered positive; samples with CT values above 40 were considered to be negative. DNA from confirmed P. falciparum samples were subsequently sequenced to examine single-nucleotide polymorphisms (SNPs) in the genes: P. falciparum chloroquine resistance transporter (pfcrt), P. falciparum multidrug resistance associated protein 1 (pfmdr1), and P. falciparum kelch13 (pfk13) using protocols from previous studies [Genomic DNA was extracted from 350 µl of blood using the EZ1 DNA blood Kit and the automated system EZ1 Advanced XL  following the manufacturer´s instructions. Samples were eluted in 100 µl and screened using the multiplex photo-induced electron transfer (PET) real-time PCR (PET-PCR) for species confirmation , 18. PET studies , 19, 20.P. falciparum neutral MS  located in chromosomes 2, 3, 4, 6, and 12 were amplified using previously published methods [P. falciparum was already established in the laboratory. Post-PCR fluorescently labeled products were separated on an Applied Biosystems 3130 capillary sequencer and scored using GeneMarker v1.95 (SoftGenetics LLC). The presence of more than one additional allele in a single locus was interpreted as a coinfection with more than one genetically distinct clone in the patient. Missing data (no amplifications) were not considered for defining haplotypes. The size of the alleles obtained in this study was normalized with 1 bp for analysis and comparison with published alleles previously.Seven  methods , 22 sincP. falciparum, 44 (67.7%) for P. vivax, 2 (3.1%) for P. ovale, and 3 (4.6%) were mixed infections  . Two samples previously identified as P. vivax by microscopy were reported as mixed  infections by PET-PCR. Follow up samples (microscopy slides) were taken for 12 patients; however; only four presented symptoms and confirmed P. vivax infections more than 28 days after the initial infection , 16 (24.6%) were positive for Anopheles breeding sites have been described  of patients were 26–40 years old, 3 (4.6%) were 5–14 years old, 11 (16.9%) were 15–25 years old, 10 (15.4%) were 41–50 years old, and 9 (13.8%) were > 50 years old; 1 patient’s age was not reported. According to the travel history, 61.7% of the positive cases came from South American countries, mainly from Brazil, Colombia, Peru, and Venezuela, 24.7% of the patients reported travels to Africa, and 13.6% of the patients traveled to Central America, Asia, and other countries  , double-mutant CVMET (codons 75–76) (Colombia/Nicaragua), double-mutant StctVMNT (codons 72–76) (Venezuela), and single-mutant CVMNT (codon 76) (Colombia) , 746,465 immigrants  live in Chile and it is estimated that this number has increased more than 300% compared to the previous decade  .Although malaria has been eliminated in Chile, globalization and cultural exchange has contributed to raising awareness of the disease and for that reason early detection of malaria cases have improved in recent years. It is necessary that health care providers in Chile obtain the travel history of patients in order to diagnose malaria promptly, notify health authorities and send samples for confirmation to the ISP. Moreover, malaria should be included in the differential diagnosis for every patient with fever who has travelled to an area where malaria is endemic .P. vivax infections. Unfortunately, complete travel history, prophylaxis, and anti-malarial treatment could not be obtained for all cases. Nevertheless, as expected, the travel history matched the infecting species expected, with P. vivax infection identified in people who visited P. vivax endemic countries in South America and Asia and P. falciparum mainly from Africa. While no clusters were detected in our analysis, a cluster of P. vivax cases was previously reported among Chilean travellers returning from Peru [In this study, the most frequent malaria cases were due to rom Peru . This hiAnopheles vector has been confirmed. The existence of imported malaria cases, which are possible reservoirs of the infection, together with the occurrence of favourable climatic conditions for competent vector species, represent a risk for the onset of autochthonous malaria cases in the country. Therefore, vigilance and continued surveillance should be warranted.In Chile, imported malaria cases have been reported in areas that were previously considered endemic and where the presence of the pfcrt gene reported in this study  are known chloroquine resistance markers found worldwide. Their frequency in endemic countries and the possible origin of each genotype is associated with different geographical locations [IET genotype is typically found in Africa, in countries such as Uganda, Guinea and Benin [StctVMNT genotype, found in samples from Venezuela, has two possible independent origins, one in South America and one in Papua New Guinea [ET genotype found in a sample from a patient with travel history to Colombia and Nicaragua is mainly reported in the northern part of South America. The CVMNT genotype identified in two samples from Colombia is the closest to the ancestral sensitive genotype, which has been previously reported in Brazil, Colombia, Ecuador and Peru. The wild-type pfcrt genotype is commonly found in Central America [Additionally, the drug resistance profiles of the parasites are important to establish the guidelines for an appropriate treatment policy. Mutations in the ocations , 27. Fornd Benin , 28–34. w Guinea , 27. The America .pfmdr1 alleles identified in the samples analyzed, showing single, triple, and quadruple mutants. Mutations at pfmdr1 N86Y and D1246Y, which are common in Africa, have been linked to decreased sensitivity to chloroquine and amodiaquine, but increased sensitivity to lumefantrine, mefloquine, and artemisinins [C and 1042D), are associated with altered sensitivity to lumefantrine, mefloquine, and artemisinins [F), which has been previously reported in these countries [P. falciparum confirmed that the samples analyzed were all imported, clusters or clonal expansions were not found. Furthermore, the haplotypes identified in this study, were all different  [An important aspect within an Integrated Surveillance System, such as the one Chile is currently implementing, is to join efforts to obtain complete epidemiological information on all cases reported, including using adequate diagnostic tools in primary health centers, following up all malaria cases, recommending appropriate prophylaxis to Chilean travellers, and ensuring the availability of appropriate drugs for malaria treatment. Currently, atovaquone–proguanil  or mefloquine could be used as the first-line treatment for patients with uncomplicated malaria in Chile . Among treports) .It is essential to strengthen the malaria surveillance system in Chile, which should include complete epidemiological data, travel history, previous diagnostic results and prophylaxis or treatment given. This information is valuable to better assist patients, provide recommendations to travellers and further analyse the current malaria situation in the country. Moreover, the introduction of malaria cases in Chile has brought the presence of resistant strains from all over the world, which requires proper control and surveillance.


Plasmodium falciparum infections in the first year of life within a birth cohort in Kintampo, Ghana (N = 1855). Infants were monitored at home with monthly sampling and also at the clinic for any febrile illness between 2008 and 2011. Light microscopy was performed on monthly scheduled visits and febrile ill visits over twelve months of follow-ups (n = 19231). Microscopy-positive visits accompanied with or without symptoms were rare during the first five months of life but were common from six to twelve months of age. Among 1264 infants with microscopy data over a minimum of eight monthly visits and also throughout in sick visits, some were microscopy negative (36%), and others positive: only-symptomatic (35%), alternating (22%) and only-asymptomatic (7%). The median age of microscopic infection was seven months for the alternating group and eight months for both the only-symptomatic and only-asymptomatic groups. The alternating group had the highest cumulative incidence of microscopic infections, the lowest age at first infection and 87 different infection patterns. Parasite densities detected by microscopy were significantly higher for symptomatic versus asymptomatic infection. We conclude that infants in malaria endemic areas experience diverse infection profiles throughout their first year of life. Further investigations should include submicroscopic reservoir and may shed more light on the factors that determine susceptibility to malaria during infancy.Although malaria mortality among children under five years of age is high, the characteristics of their infection patterns are not well described. The aim of this study was to examine the longitudinal sequence pattern of  Plasmodium falciparum malaria is often uncomplicated, but can also be fatal, and children below the age of five are most vulnerable  and only-asymptomatic groups [1.85 visits/child (161/87)] (p = 0.715). Within the alternating group, the mean number of microscopy positive visits that were with symptoms was 2.17 infections/child (595/274) and without symptoms was 1.86 infections/child (509/274) (p < 0.001).When comparing infants with two or more microscopic infections, the alternating group had the highest mean number of infections [4.03 infections/child (1104/274)] compared to the only-symptomatic [2.69 infections/child (533/198)] and only-asymptomatic groups [3.18 infections/child (108/34)] (p = 0.001).Analysis of the number as well as the sequence patterns of microscopic infections in the alternating group showed 87 different sequences of symptomatic malaria (S) and asymptomatic microscopic infections (A) . Half 5.An analysis of the number of visits in which microscopy positivity was with or without symptoms within the alternating group showed a maximum of ten intermittent microscopic infections, while three microscopic infections were frequent [observed among 25% (68/274) of infants in six different sequence patterns] . For theThe time span  between any two visits in which microscopy was positive peaked between 27 and 38 days for all groups. Visits in which microscopy positivity were six months (180 days) or more apart were rarely observed in all groups, while positivity which were less than 27 days apart were rare in the only-asymptomatic group .The first visits in which microscopy was found positive were between one and twelve months of age . The age at first visit where microscopy was positive was lowest in the alternating group [median = 5 months (IQR: 4–7)] compared to the only-asymptomatic group [median = 7 months (IQR: 4–9)] or the only-symptomatic group [median = 7 months (IQR: 5–9)] .Whereas the first symptomatic malaria episodes were detected between two and twelve months of age for infants in the only-symptomatic group , the firAlthough microscopic infections were detected at all ages, a relatively small proportion of infants  had visits in which microscopy was positive at two months of age and were mainly in the alternating group [69% (25/36)]. For all the groups with malaria parasites, the number of times that infants had visits in which microscopy was positive (on separate occasions) increased with age, but the proportion of infected infants decreased with age . The medP. falciparum parasite density was 2–974759 parasite per μl. Parasite density increased with age in all the groups ] was higher when infants were symptomatic compared to when they were asymptomatic [2848 parasite/μl (IQR: 858–8228)] (p < 0.001). In all groups, the parasite density was lower in the first six months compared to seven months of age and above .The longitudinal profiles and age-specific patterns of microscopy positivity with or without symptoms in 19231 monthly and ill visits were examined in a birth cohort of 1819 infants from a high malaria transmission area of Ghana. Despite living in the same area during the first year of life, infants experienced highly diverse patterns of microscopy positivity. Four main groups of infants were identified, namely: parasite negative (36%), only-symptomatic (35%), alternating (22%) and only-asymptomatic (7%). Over half of the 87 different patterns of alternating symptomatic malaria and asymptomatic microscopic infection were unique to individuals, indicating no uniform sequence of alternating symptomatic and asymptomatic microscopic infections.In unscheduled/clinical visits where microscopy positivity was with symptoms, microscopy was performed on the same day and infants were treated, while on scheduled/home visits when infants were ‘healthy’, microscopy was performed at a later date and there was no intention to treat at the visit. Nevertheless, all visits where microscopy positivity was with or without symptoms (asymptomatic microscopic infections) were intermittent, with the asymptomatic microscopic infections spontaneously clearing up in the only-asymptomatic group. Given the gradual development of naturally acquired immunity to malaria at intermittent exposures , 31 and The higher cumulative incidence of symptomatic and asymptomatic microscopy positive visits in the alternating group suggests that in vivo or extrinsic protection against infections may be relatively poor for infants in this group. Importantly, the only-asymptomatic or only-symptomatic patterns of infection may be observed less frequently among infants in high transmission areas, compared to the alternating pattern, reducing their detection and significance to malaria in the first year of life. For instance, microscopic infections which were asymptomatic, were frequently followed by symptomatic malaria  among children aged six months to five years of age, in urban Uganda . ProspecThe age at first microscopy positivity was lower for the alternating group (seven months) than for both only-asymptomatic and only-symptomatic groups (eight months). However, Wagner and others  reportedConsistent with the classic age distribution of infections in infants , 37, 38,P. falciparum (96%), P. malariae (2%) and P. ovale (1.4%) was similar to that observed in other malaria endemic areas of Africa [The frequency of the asexual stages of f Africa . The sexf Africa , 41.Generally, studies in malaria endemic areas detect low parasite density in both asymptomatic infections and symptomatic malaria in infants below six months of age , 13. SimIn this birth cohort study, we had access to a large set of microscopy data, including the parasite density, among infants living in a high transmission area. We did not assess the substantial proportion of submicroscopic infections (may be symptomatic or asymptomatic) that can be detected using more sensitive molecular tools such as PCR , 42. We Despite its limited sensitivity compared to PCR, it is worth noting that microscopy has been extensively used to learn about the etiology, epidemiology and dynamics of malaria; it is considered a gold standard and is a more helpful method for detecting and managing clinical cases as described in the WHO malaria management guidelines , 43. StaThe sampling interval is important for monitoring the incidence of asymptomatic carriage and our study is one of few studies that have sampled young children monthly and even provided additional samples during ill-visits which occurred in-between the monthly sampling –53. TypiIn view of these limitations and strengths, it may be necessary to agree unanimously on definitions which consider sampling intervals for various malaria exposure groups and transmission settings, to reduce the under-reporting of asymptomatic infections. Further, intervention studies aimed at reducing malaria transmission may need to ascertain the entire burden of asymptomatic infections, including those that are submicroscopic, for optimal impact. In summary, this study’s limitation lies primarily in the use of microscopy detection methods, while the strength lies in the frequency of sampling and the analyses of a large number of monthly samples over the first twelve months of life.Given the observed complex heterogeneity of microscopic infection patterns during infancy, studies based on single/few cross-sectional samples may briefly capture the effects of malaria exposure and consequently restrict the understanding of susceptibility and immunity to malaria in infancy. Grouping infants on the bases of their temporal malaria infections may help to better understand the immunological mechanisms of protection against malaria. The results potentially open up new opportunities for the improved analyses of host  and external determinants , which can guide future studies designed to control and eliminate malaria in endemic areas.In conclusion, within the same transmission area, we detected by microscopy groups of infants that remained either parasite-free or with only asymptomatic infections or symptomatic malaria alone, or that intermittently alternated between asymptomatic infections and symptomatic malaria, through their first year life. These groups of infants were significantly different per their malaria parasite density and age at microscopy positivity. In-depth investigations involving the submicroscopic reservoir, to identify the underlying determinants of the variation between the groups of infants, can contribute to the understanding of malaria susceptibility during infancy.S1 Fig(PDF)Click here for additional data file.S2 Fig(PDF)Click here for additional data file.S3 Fig(PDF)Click here for additional data file.S4 Fig(PDF)Click here for additional data file.S1 Table(PDF)Click here for additional data file.


P. falciparum in pregnant women.In low transmission settings early diagnosis is the main strategy to reduce adverse outcomes of malaria in pregnancy; however, microscopy and rapid diagnostic tests (RDTs) are inadequate for detecting low-density infections. We studied the performance of the highly sensitive-RDT (hsRDT) and the loop mediated isothermal DNA amplification (LAMP) for the detection of P. falciparum in peripheral blood, using a quantitative reverse transcription PCR (qRT-PCR) as the reference standard. Diagnostic performance of hsRDT and LAMP were compared with routine testing.A cross-sectional study was conducted in two malaria-endemic municipalities in Colombia. We screened pregnant women in the context of an antenatal care program in health facilities and evaluated five tests  for the detection of P. falciparum was 4.5% by qRT-PCR, half of those infections were subpatent. The sensitivity of the hsRDT (64.1%) was slightly better compared to microscopy and cRDT (59 and 53.8% respectively). LAMP had the highest sensitivity (89.7%) for detecting P. falciparum and the ability to detect very low-density infections (minimum parasite density detected 0.08 p/μL).The prevalence of Plasmodium spp. infections by tests routinely used in pregnant women attending antenatal care visits. LAMP methodology can be successfully implemented at local hospitals in malaria-endemic areas. The relevance of detecting and treating this sub-patent P. falciparum infections in pregnant women should be evaluated.There is an underestimation of NCT03172221, Date of registration: May 29, 2017.ClinicalTrials.gov, Identifier:  Plasmodium infection and malaria in pregnancy is associated with well-documented adverse outcomes for both the mother and foetus; the main deleterious effects include maternal anemia, miscarriage, premature delivery and low birth weight [Plasmodium falciparum [. Since the overall transmission intensity in the continent is relatively low, intermittent preventive treatment during pregnancy is not recommended and the main strategies to limit the burden and consequences of malaria are based on “prompt diagnosis and effective treatment” combined with the use of long-lasting insecticide treated bed nets [P. falciparum parasites to massively sequester in the placenta also contributes to a reduced detectability of maternal infections in peripheral blood [Pregnant women are especially susceptible to h weight –4. In thlciparum . Since tbed nets , 7. In Cbed nets . Howeverbed nets . Moreoveal blood , 9.Although microscopy remains the mainstay of malaria diagnosis in many endemic settings, this method provides limited sensitivity and requires well-trained personnel as well as adequate laboratory reagents and equipment. In Colombia, microscopy-based diagnosis has been shown to miss between 20 and 75% of maternal infections detected in peripheral blood by Polymerase Chain Reaction (PCR) –15. RDTsMore sensitive diagnostic tools are needed for an accurate identification of infections. Molecular methods based on nucleic acid amplification techniques, such as PCR, can detect very low parasite densities, but are generally impractical for wide-scale clinical use as they rely on sophisticated equipment and highly-skilled staff, which are rarely available in most malaria endemic settings , 22. ManP. falciparum histidine rich protein 2 (HRP2) is available since 2017 . This “highly sensitive” RDT (hsRDT) showed an improved sensitivity as compared to conventional RDTs (cRDT) when using PCR as reference standard in samples collected from asymptomatic individuals, suggesting that the new test could be a useful tool for malaria elimination strategies [A malaria RDT with a significantly improved sensitivity for the detection of rategies –26. In frategies .P. falciparum infections when compared to microscopy (77.1%) and cRDTs (77.1–82.8%), using nested PCR (nPCR) as reference standard [P. falciparum during routine antenatal care visits in two municipalities of Colombia. In addition, the results were compared to the performance of other standards of practice such as the microscopy or the cRDTs.By combining a significantly improved sensitivity together with the ease-of-use and affordability of cRDT, the hsRDT represents a potentially useful tool to identify the especially hard-to-detect malaria infections in pregnant women. In a previous retrospective study using peripheral and placental specimens collected in Colombian pregnant women, the hsRDT showed a slightly improved sensitivity (85.7%) for detecting standard . The difstandard . Howeverstandard . This stP. falciparum is the predominant species in both sites, representing 70 and 90% of all malaria cases, respectively [A cross-sectional study was conducted in two endemic municipalities from Colombia, between August 2017 and January 2018. The two municipalities are considered areas of high risk of infection, as defined by annual parasite incidence (number of cases per 1000 inhabitants) of 100.8 in Quibdó (2016) and 16 in Tumaco (2016). ectively . It is eectively .P. falciparum parasites mutant for the pfhrp2 gene have been reported in the Amazonas Department , southern Colombia. These parasites were also mutant for the pfhrp3 gene, which make them undetectable by HRP2-based malaria RDTs. However, the prevalence of this genetic deletions is rare in other endemic regions of Colombia, including where this study was conducted [In Colombia, onducted .Pregnant women, self-presenting at health centers for antenatal care visits and who met the following inclusion criteria were included: confirmed pregnancy, registered to the antenatal care program in the local health center, ≥15 years old and resident for at least 1 year in the study site. The following exclusion criteria were considered: past history of malaria or antimalarial drugs in the last 3 months, having tested positive for malaria by microscopy or cRDT in any previous study visit, symptoms and signs of severe malaria as defined by WHO. Malaria-infected pregnant women were treated based on results of diagnostic tests accepted locally – i.e. microscopy and cRDT – and according to local policy.The sample size was calculated according to a previously published methodology  to obtaiAll study participants provided a written informed consent or informed assent  to participate in the study. The study protocol and documents were reviewed and approved by the ethics committee of Medical Faculty-Universidad de Antioquia (Record 010 of 22-05-2017). Additional consent was also obtained for future use of biological samples. For women younger than 18 years old, additional written approval from the parent or legal guardian was required. The study was conducted in accordance with the Declaration of Helsinki and local rules and regulations of Colombia.Each participant provided 290 μL of finger-prick blood that was handled as follows: 10 μL for cRDT and hsRDT (5 μL for each test); 20 μL for microscopy; 60 μL for DNA extraction and LAMP; 100 μL spotted on filter papers (Whatman 3) for DNA extraction and nPCR; and 100 μL spotted on filter papers (Whatman 903) for RNA extraction and qRT-PCR. Testing by microscopy, cRDT , hsRDT  and LAMP  were performed in field conditions in the local hospitals.Testing by microscopy, cRDT , hsRDT  and LAMP  were performed in field conditions and conducted in the public facilities of local hospitals. The cRDT and hsRDT were performed by community health workers, microscopy by a malaria microscopist and LAMP by a clinical microbiologist. The study staff received appropriate training before the study initiation.The hsRDT and cRDT were performed according to the manufacturer’s instructions. The test results were read at 15 min for cRDT by one independent reader and at 20 min for hsRDT by two independent readers blinded to each other. The results were interpreted as invalid (no control line or red background), positive (control line and test line), or negative .Microscopy was performed according to the WHO guidelines . A smearPlasmodium spp (Pan-LAMP) and a P. falciparum specific (Pf-LAMP) LAMP assays amplifying Plasmodium mitochondrial DNA were used according to the manufacturer’s instructions. All Pan-LAMP positive samples were subsequently tested using Pf-LAMP. Results were read by visual fluorescence and classified as negative (Pan-LAMP negative), P. falciparum positive (Pan-LAMP and Pf-LAMP positive) or Non-falciparum positive (Pan-LAMP positive and Pf-LAMP negative).For LAMP, DNA extraction was performed using the boil and spin method as described elsewhere . A combiPlasmodium spp amplification followed by a nested species-specific PCR . DNA was extracted from blood-spot filter using QIAamp DNA Mini Kit , according to the manufacturer’s instructions. The limit of detection of the nPCR was estimated at approximately 1 p/μL.A nPCR, based on the amplification of the 18S rRNA gene, was performed as previously described . BrieflyPlasmodium 18S rRNA, was performed at the University of Washington as previously described and was used as reference [P. falciparum 18S rRNA added to parasite-free whole blood. Copies/mL of whole blood was used to estimate p/μL of whole blood using a conversion factor  and classification as P. falciparum or non-P. falciparum infections.A qRT-PCR based on the amplification of eference , 36. Brieference  and thenFebrile women were those presenting fever (≥37.5 °C axillary temperature) at enrolment or with a self-reported history of fever within the 3 days preceding enrolment . AfebrilDemographic, clinical, obstetrical information and test outcome was collected using case report form and double entered in Open Clinica system. Descriptive analysis was carried out using SPSS version 25 software and Excel (Microsoft). Test performance analyses were conducted with STATA version 11 software . The Mann-Whitney test was used for comparison of mean parasite densities. Graphs were prepared with Graphpad Prism 8.1. , except for the Venn diagrams that were generated with the InteractiVenn tool .A total of 991 pregnant women were screened and 880 (89%) were enrolled  non-P. falciparum and 1 (2%) mixed infection. Subpatent infections (n = 24) represented 42% (16/38) of P. falciparum infections and 100% (8/8) of non-P. falciparum ones.A total of 47 out of 858 (5.5%) CR Table , includin = 9.2 p/μL) infections, as compared to patent  and febrile (geo. Mean = 270 p/μL) infections . Likewise, infections detected during the first trimester of pregnancy showed higher densities than those occurring in the second or third trimester, but the difference did not reach statistical significance (p = 0.053)  with a geometric mean of 13.2 p/μL. The parasite density was significantly lower in subpatent (geo. Mean = 0.38 p/μL) and afebrile . This translated in 64.1% (95% CI: 47.2–78.8) sensitivity and 99.9% (95% CI: 99.3–100.0) specificity for the hsRDT, and 89.7% (95% CI: 75.8–97.1) sensitivity and 99.9% (95% CI: 99.3–100.0) specificity for LAMP. The microscopy, cRDT, and nPCR comparator tests identified respectively 23, 21, and 30 of the 39 P. falciparum infections, resulting in sensitivity of 59% (95 CI: 42.1–74.4), 53.8% (95 CI: 37.2–67.9), and 76.9% (95CI: 60.7–88.9). All the 819 negative samples were correctly identified as such by these tests, resulting in 100% specificity  for each of them. The PPV and NPV estimates were > 96% for all tests evaluated in this study. The hsRDT detected 22% (4/18) of the positive samples missed by the cRDT . Although the hsRDT had higher sensitivity than microscopy and cRDT, the superiority was not established . Microscopy, cRDT and hsRDT performed poorly below 10 p/μL. However, while the lowest parasitemia detected by the standards of practice (microscopy and cRDT) was 13.1 p/μL and 42.3 p/μL, respectively, the hsRDT was able to identify all the infections over 1.8 p/μL. LAMP had the best performance across low density ranges and was able to detect 75% of the infections below 1 p/μL (highest undetected parasitemia was 1.56 p/μL)  ranged from 0.89 to 27.8 p/μL  ranged from 0.08 to 1.80 p/μL , overall results showed that conventional diagnostics tests like microscopy and cRDT were unable to detect a large proportion of asymptomatic and low-density infections. LAMP had the highest sensitivity for detecting P. falciparum in pregnant women, and both tests missed a higher number of low-density infections as compared to PCR [Plasmodium infections by tests routinely used in pregnant women attending antenatal care visits.Malaria prevalence in pregnant women was found to be of 5.5% by qRT-PCR, similar to previous studies conducted in Colombia , 14, 15,d to PCR , 20, 38.P. falciparum infections in peripheral samples from pregnant women (64.1%) was slightly better than that of routinely used tests during antenatal care visits (59% by microscopy and 53.8% by cRDT). Similar findings have been reported in pregnant women [P. falciparum–positive pregnant women detected by hsRDT but not by cRDT result in a gain of 10% in diagnostic sensitivity, however, the hsRDT was not superior to cRDT. The additional infections detected by hsRDT but not by cRDT had parasite densities ranging from 0.9 to 27.8 p/μL, indicating that hsRDT would confer a meaningful improvement detecting low density infections. The relevance of detecting and treating these additional P. falciparum infections in pregnant women should be evaluated [The sensitivity of the hsRDT to detect nt women , 24, 25 nt women . The fouvaluated .P. falciparum infections were observed among the tests when compared to qRT-PCR as the reference standard. LAMP showing the highest sensitivity (89.7%) and ability to detect very low-density infections (minimum parasite density detected 0.08 p/μL). These results were consistent with previous studies proving higher performance of LAMP for detecting maternal Plasmodium infection [P. falciparum mutant for the hrp2/hrp3 genes and non-P. falciparum malaria infections.Differences in the sensitivity to detect nfection , 23. In Plasmodium infections during pregnancy, including low density infections should be studied, as well, the relevance of pregnant women as sentinel population for epidemiological surveillance and the role of pregnant women as transmission reservoir.All the evaluated tests showed an improved performance for first-trimester testing (> 85% sensitivity) compared to second and third trimester, but especially LAMP which was able to detect all infections in the first trimester. This finding is important due to the relevance of early stage detection to prevent deleterious effects of malaria in pregnancy . OverallP. falciparum infections, could make the case for a practical and cost-effective use of LAMP in the frame of integrated maternal and child health services. Yet, further studies are needed to strengthen the relevance of detecting and treating cRDT−/microscopy-negative infections and to guide recommendations on the use of LAMP and hsRDT in pregnant women with inherently low-density P. falciparum levels. A validation in different transmission settings and the cost-effectiveness of integrating new tools in the maternal health programs should also be evaluated.LAMP is significantly simpler to implement than some of the more complex molecular methods but still requires several sample processing steps to be conducted and currently retails at a higher cost-per-test than cRDT and hsRDT. The fact that antenatal care is typically provided in clinics where the basic laboratory procedures required by LAMP can be done, together with the fact that there might be a significant direct and long-term clinical benefit in detecting and treating low density Plasmodium spp. infections by tests routinely used in pregnant women attending antenatal care visits, like microscopy and RDTs. The sensitivity of the hsRDT to detect P. falciparum infections in peripheral samples was slightly better than that of routine used tests, however, a significant proportion of infections (38.5%) remained undetected by cRDT, microscopy, or hsRDT. LAMP showed the highest sensitivity and ability to detect very low-density infections and their methodology can be successfully implemented at local hospitals in malaria-endemic areas. The relevance of detecting and treating this sub-patent P. falciparum infections in pregnant women should be evaluated, to guide recommendations on the use of LAMP and hsRDT.There is an underestimation of Additional file 1: Figure S1. Distribution of P. falciparum densities stratified by maternal characteristics. P. falciparum mono-infection parasite densities  estimated by RT-qPCR. (A) Distribution of parasite density among 38 qRT-PCR positive pregnant women. (B-E) Mean of parasite density stratified by maternal characteristics. Horizontal bar indicates the geometrical mean. Subpatent: Infection detected by qRT-PCR, but not detected by LM or cRDT. Febrile: Fever at enrolment or reported fever on the last 3 days .Additional file 2: Figure S2. Superiority and equivalence of the hsRDT for the detection of P. falciparum during pregnancy compared to other diagnostic tests. A) Superiority testing between hsRDT and Microscopy/cRDT. Confidence Intervals (CI) of the difference between sensitivities compared to a superiority margin of + 15%. *One-sided comparative test not significant: p-value> 0.05. Dot: difference; bar: confidence interval; blue: area above the superiority margin. B) Equivalence testing between hsRDT, LAMP and nPCR. CIs of the difference between sensitivities compared to an equivalence margin of ±3%. * Two one-sided test (TOST) for equivalence not significant: p-value> 0.05. Dot: difference; thick bar: 90% CI; thin bar: 95% CI; green: area within the equivalence interval. cRDT ; hsRDT (highly sensitive Rapid Diagnostic Test); nPCR (nested Polymerase Chain reaction), LAMP , qRT-PCR (Quantitative Reverse Transcription polymerase chain reaction).Additional file 3: Table S1. Comparison of diagnostic test performance according to febrile status, maternal age, gravidity and pregnancy trimester of the study participants.