Plasmodium falciparum prevalence at baseline and 10 weeks post-campaign were similar: 1.31% versus 1.43%, respectively. Prevalence of serological markers were similar at 10 weeks post-campaign compared with baseline, and increased at 6 months. No severe adverse events associated with the MDA were identified in the pilot; there were severe adverse events in a separate, subsequent campaign. Both MDA and IRS are acceptable and feasible interventions in Haiti. Although a significant impact of a single round of MDA/IRS on malaria transmission was not found using a standard pre- and post-intervention comparison, it is possible there was blunting of the peak transmission. Seasonal malaria transmission patterns, suboptimal IRS coverage, and low baseline parasitemia may have limited the effectiveness or the ability to measure effectiveness.For a malaria elimination strategy, Haiti’s National Malaria Control Program piloted a mass drug administration (MDA) with indoor residual spraying (IRS) in 12 high-transmission areas across five communes after implementing community case management and strengthened surveillance. The MDA distributed sulfadoxine–pyrimethamine and single low-dose primaquine to eligible residents during house visits. The IRS campaign applied pirimiphos–methyl insecticide on walls of eligible houses. Pre- and post-campaign cross-sectional surveys were conducted to assess acceptability, feasibility, drug safety, and effectiveness of the combined interventions. Stated acceptability for MDA before the campaign was 99.2%; MDA coverage estimated at 10 weeks post-campaign was 89.6%. Similarly, stated acceptability of IRS at baseline was 99.9%; however, household IRS coverage was 48.9% because of the high number of ineligible houses. Effectiveness measured by  Plasmodium falciparum, with 39,097 cases of P. falciparum malaria reported in 2020.,,,,The Republic of Haiti, along with the bordering country of the Dominican Republic, are the only countries in the Caribbean with endemic malaria transmission. The two countries  comprise the island of Hispaniola and report malaria caused almost exclusively by –In the past decade, MDA received renewed interest from the global malaria community and donors. Multiple randomized, controlled trials have found that MDA rapidly reduces parasite prevalence, and thus could be useful in accelerating malaria elimination in low-transmission countries.P. falciparum parasite to this drug. Haiti is one of the few countries where the P. falciparum parasite remains susceptible to SP.No recent MDA campaign has used solely a single-dose treatment for malaria elimination because most antimalarial medications require a multiple-day regimen. Although sulfadoxine–pyrimethamine (SP) is administered as a one-dose treatment, there is widespread resistance of the In line with the WHO’s recommendation, the MDA/IRS campaign was implemented in 2018 along with strengthening malaria surveillance, case management, and entomological monitoring capacity. This article focuses on the results of the baseline and three follow-up surveys for the MDA/IRS component of the package of interventions. The study was part of the Malaria Zero project to support Haiti and the Dominican Republic in accelerating elimination of malaria from the island of Hispaniola.This pilot was conducted in the Grand’Anse Department located on the southern peninsula of Haiti. The MDA/IRS campaign was implemented in selected communities of five communes  in the Grand’Anse Department, where typically > 50% of Haiti’s malaria cases have been reported.,Bacillus thuringiensis israelensis) application (December 2018). In addition, an LLIN campaign was conducted in Grand’Anse in October 2017 under Haiti’s GF grant to distribute two LLINs per household through community fixed posts.Routine malaria control program interventions have been implemented nationally under Haiti’s Global Fund (GF) malaria grants since 2003. The GF grants have supported aggregated malaria case reporting through the national surveillance system, free access to malaria testing with histidine-rich protein 2-based (HRP2) rapid diagnostic tests (RDTs), treatment with CQ plus a single dose of primaquine PQ, larval source management, and the distribution of long-lasting insecticide-treated nets (LLINs).2). In July 2018, all residents in the study area were censused and houses were georeferenced. The census provided the sampling frame for households. The study used an observational, cross-sectional design to assess parasite prevalence, malaria seroprevalence, and sociobehavioral factors before the MDA/IRS campaign (baseline) and then post-campaign planned at 6 weeks, 6 months, and 12 months. (The first and third post-campaign surveys were delayed and are described in “Results.”) The time points for the surveys were selected for the following reasons: 1) at baseline (T0) data collection scheduled at the beginning of malaria transmission season before the MDA/IRS campaign, 2) at 6 weeks (T1) to evaluate the effect of the campaign that is expected within 1 to 3 months, 3) at 6 months (T2) to evaluate any extended effect during the low-transmission season and to assess seroprevalence changes using short- and mid-term antibody markers, and 4) at 12 months (T3) to obtain a comparison at the same transmission season as baseline and assess seroprevalence changes using longer term antibody markers.Communities with high malaria transmission (relative to other communities in Haiti) were identified based on surveillance data, modeled predictions of malaria transmission [malaria basic reproductive rate under control (Rc)], and a rapid assessment of parasite prevalence and seroprevalence, which have been described elsewhereThe sample size per survey was calculated using the following parameters: 80% statistical power, 5% chance of a type I error (two sided), and an intracluster correlation coefficient of 0.1277 based on prior study data. A sample size of 1,250 individuals for each survey would detect a 15% decrease in parasite prevalence from baseline to the follow-up time point. Based on previous surveys, the average household size was expected to be approximately 3.5 persons. With an estimated survey refusal rate of at least 4%, 35 households were targeted for sampling per OU.P. falciparum parasite susceptibility profile in Haiti, the global experience with the use of SP in seasonal malaria chemoprophylaxis campaigns, intermittent preventive treatment in pregnant women, preferred operational aspects for mass administration (single-dose regimen), and consensus across partners, with the final decision made by the Haitian Ministry of Health. An SLD of 0.25 mg/kg PQ  was included in the MDA regimen for the gametocytocidal effect to decrease parasite transmission from existing infections at the time of the campaign.–The intervention was a combined house-to-house MDA and IRS campaign in communities with a total population of 40,019 people. Both interventions were preceded by community engagement activities. From September 3 to 14, 2018, training sessions were conducted on malaria interventions and their health benefits with Community Health Councils (CHCs), which were voluntary community leadership groups organized as part of the Malaria Zero project. Prior to and during the MDA and IRS campaigns, CHCs mobilized their communities during weekly outreach activities using microphones, church and school meetings, door-to-door outreach, and public announcements. Radio broadcasts ran from August 28 to November 15, 2018 on nine local stations, banners were set up in high-visibility locations, and sound trucks were deployed just prior to each campaign. Campaign staff worked with the CHCs throughout the campaign to conduct outreach and to address community questions and concerns. The MDA component of the campaign was implemented from October 10 to November 6, 2018 and was coordinated with IRS implementation. The selection of SP for the MDA was based on the Pharmacovigilance (PV) using the WHO definitions for monitoring and reporting adverse events (AEs) was initiated during the first week of the MDA component of the campaign.Anopheles albimanus, tends to be exophagic and exophilic, these characteristics are not exclusive.A. albimanus population.,Although the main malaria vector, All residents of the sampled households were provided information on the risks and benefits of participating in the survey before seeking consent. Adults (≥ 18 years old) who consented were eligible to participate. Children and minors (< 18 years old) were eligible if parents or guardians consented. In addition, assent was sought from minors (7–17 years old). If family members were absent during the first visit, up to two additional visits to the household were made within a 3-day window to meet the absent members.Standardized survey questionnaires were programmed into the CommCare platform and loaded onto tablets. The residence location, GPS coordinates, altitude, and household member composition were collected. Household-level questions were posed to the head of household or primary caregiver to assess attitudes and acceptability toward MDA, IRS, and the goal of malaria elimination. Additional variables collected at the household level included operational aspects of the intervention delivery by campaign teams, awareness of the campaign, and household-level ownership of LLINs. Individual-level variables captured demographics, whether the medications were ingested, attitude toward MDA, AEs, and reasons for not taking the MDA medication.P. falciparum parasites by a conventional RDT  and a highly sensitive RDT (hsRDT) , and to prepare dried blood spots on filter papers . If either the RDT or hsRDT was positive, the malaria parasitemia status was considered positive for the analysis. Studies show a modest advantage of hsRDTs for detecting low-density parasitemia compared with RDTs, especially in asymptomatic populations and could aid in identifying more infections.Plasmodium. Detection of HRP2 antigen by multiplex laboratory assay was performed as described previously.,P. falciparum exposure, whereas Etramp 5 ag 1 was selected to represent recent (6 months) infection with P. falciparum.,,A finger-prick blood sample was collected from each consenting participant to test for the presence of Multiple data sources were used in this analysis. The primary data source was the pre- and post-campaign household surveys with the collection of biomarkers. Additional data sources included the campaign monitoring data, PV reports of AEs associated with the MDA campaign, the pre-survey census, MDA and IRS campaign monitoring data, and insecticide wall bioassays. Analyses, accounting for clustering at the OU level, were conducted in SAS version 9.4 . For calculating point estimates and their corresponding 95% confidence limit (CL), sampling weights were based on the probability of selecting the household in each of the 12 OUs. Empirically estimated SEs were used to account for the correlation of individual data within households.Main characteristics of the baseline and three follow-up surveys are presented in Prior to the campaign, the stated acceptability of MDA and IRS was high at T0, when the primary respondents of the households were asked whether they would take medications or allow spraying in their home for malaria elimination—nearly all responded yes . This reflected the expected willingness of the head of household or primary caregiver to accept the interventions delivered by the campaign, and thereby reach individual household members .The acceptance of the MDA medications during the campaign was extremely high, at 95.8% of all individuals who agreed to be screened for MDA eligibility . The accThe coverage indicators were obtained at T1 to minimize the risk of recall bias. The MDA campaign coverage was high at 89.6% among all respondents of the selected households . NotablyWhen asked at T1, 48.9% of primary respondents of households stated their house had received IRS during the campaign. The combined coverage indicator for households with at least one person who took the MDA medication and had their house sprayed was 42.6%. The lower coverage of IRS reflected the percentage of structures constructed of materials suitable for IRS and drove the estimate for the combined indicator. Repeat estimates of the MDA and IRS coverage indicators at T2 and T3 were not statistically different compared with T1 .In the inland OU 14, where LLINs were distributed immediately after T1 instead of IRS, the household ownership of at least one LLIN increased from a baseline (T0) of 3.2% to 97.2% at T2, which was approximately 3 months after LLIN distribution. At T3, the household LLIN coverage fell to 34.4%, which was the same as the coverage across all study areas, highlighting the rapid loss of LLINs by 11 months after distribution .Apart from individuals’ acceptance of the campaign interventions, other factors affecting the operational feasibility of reaching high coverage were assessed. The percentage of households reached successfully to receive the interventions was an indicator of feasibility. When asked at T1, 94.8% of primary respondents stated that a team visited their house to offer MDA, whereas the remainder did not receive a visit by the team or did not know. Among 134 individuals  who did not take the medications, 61.2% responded they were absent, 17.9% were ineligible/excluded based on the MDA criteria, 14.9% refused the intervention because of personal attitudes related to the intervention, 4.5% refused based on a misunderstanding of the medical exclusions, and 1.5% did not provide a reason .For IRS, 63.5% of primary respondents indicated that a team visited their house and offered IRS. The repeat estimates at T2 and T3 revealed similar results. The suboptimal reach for IRS was a result of the high percentage of houses constructed of materials incompatible with IRS application; 36.4% of houses had walls made of cloth, plastic, or other non-sprayable (unsuitable) material. Of the 9,497 houses that were eligible for IRS, 788 (8.3%) were not sprayed. Of the 788 eligible houses not sprayed, the three main reasons included 33% of households refused IRS, 17.6% could not vacate an ill family member, and 16.5% had a head of household who was not available for consent .n = 3) were classified as “definite relationship,” whereas most other AE cases were “possible temporal relationship.” Of the 40 reports with completed data fields for symptom severity, 60% were classified as “mild” in severity (does not interfere with daily activities), 37.5% as “moderate” (may interfere with daily activities), and one AE was classified as “severe” . Upon further investigation, the severe AE was thought to be caused by infectious gastroenteritis, not the MDA medications. There were no reports of hospitalizations, clinical hemolysis , severe rash, or Stevens–Johnson syndrome during the protocol-designated PV period.Pharmacovigilance conducted between October 9 and December 6, 2018 identified 54 individuals who sought care at health facilities for potential AEs associated with the MDA . Of 16 wWhen surveyed at T1 after the campaign, 106 of 1,183 survey respondents (9.1%) who took the MDA medications reported experiencing side effects . Of the P. falciparum. See Based on the expected prophylactic duration of 30 days for SP in preventing malaria parasitemia, the primary indicator for effectiveness by parasite prevalence was assessed at the earliest follow-up survey time point, T1.At T1, there was no significant difference in parasite prevalence by RDT (1.43% at T0 versus 1.31% at T1) or HRP2 concentration detected by multiplex assay compared with the baseline, T0 . The treP. falciparum markers for all survey time points was able to approximate short-term exposure to the parasite in the past 6 months (Etramp 5 ag 1) or long-term exposure to the parasite within the past few years (AMA1/MSP1). The results for Etramp 5 ag 1 at T1 compared with T0 show an increase in seroprevalence from 10.88% to 14.44%, although the difference does not reach statistical significance. The trend for Etramp 5 ag 1 continues to increase to 18.05% at T2, which reflects the cumulative P. falciparum exposure, including the recent malaria transmission season. By the next malaria high-transmission season, corresponding to T3, Etramp 5 ag 1 decreased to 11.84%—a level similar to baseline. The longer term antibody responses marked by AMA1/MSP1 show a much higher seroprevalence level , but a similar trend as seen with the other biomarkers. However, AMA1/MSP1 estimates are within a narrow range and differences between time points are not significant  were absent or did not live there during the campaign. The campaign teams attempted to schedule return visits to provide treatment to anyone traveling and who would return home within 30 days. If capturing those who were not present  for MDA is needed to reach elimination, a strategy that uses community health workers to provide treatments to returning travelers and new residents could be beneficial. A targeted approach to reach an estimated 2,547 missed individuals (survey data) would likely be more cost-effective than another MDA round and would potentially prevent a community’s fatigue with the program.As expected, there were more challenges to the IRS intervention related to feasibility. The inland OU 14 was not amenable to IRS because of the rugged terrain and remote location. Instead, LLINs were distributed to each household, which boosted household net ownership dramatically. Our experience demonstrated that LLIN distribution is a feasible option when IRS cannot be implemented. However, the LLIN distribution was delayed by approximately 1 month because of the added complexity of coordinating an additional small campaign and exceeding the absorptive capacity of our teams and partners. Another concern regarding the incorporation of LLINs was the rapid attrition (decrease of 62.8% for households that own any bed net) by 11 months after distribution. The factors contributing to this decrease are unclear; however, it should be noted that the sample size was relatively small, resulting in a wide CI for the estimate of 34.4% household ownership at T3 . A study from EthiopiaOur pilot campaign deployed IRS as the primary, targeted vector control intervention that was expected to provide additional protection because most houses received LLINs during a 2017 mass LLIN distribution. In addition, IRS was selected for the intervention because it offered the advantages of less reliance on daily human behavior and the potential benefit of a larger excito-repellency effect against mosquitoes.–As a standard component of an MDA campaign, our pilot study implemented a passive PV system based at existing health facilities with their available staff. Both SP and PQ have been used in Haiti for many years and are generally considered to be well tolerated, especially at the limited dosing regimen  administered during the MDA. The main AEs of concern with SP and SLD PQ are severe cutaneous adverse reactions (SCARs) and acute hemolysis, respectively.P. falciparum blood-stage infection, HRP2 can persist in the blood typically up to 2 weeks after successful antimalarial treatment.The effectiveness results are intended to be interpreted in the context of what is known from randomized, controlled trials of MDA—that in areas with low to very low endemicity, a short-term reduction in parasite prevalence has been found within the initial 3 months, but thereafter there was no evidence of a sustained effect.A notable limitation of our study was the lack of a nonintervention, contemporaneous comparison group to assess effectiveness. Our pilot study was not intended to be a controlled trial because numerous randomized, controlled trials have been completed.In summary, our pilot study showed that an MDA/IRS campaign in Haiti is an acceptable and feasible strategy to implement under prevailing conditions. The use of a single-encounter medication regimen (SP and SLD PQ) likely contributed to the high coverage of MDA, whereas IRS coverage was limited by the number of houses that could not be sprayed. The distribution of LLINs instead of IRS offered logistical advantages; however, repeat LLIN distribution is needed with each MDA campaign to mitigate an expected decrease in LLIN coverage between 4 and 11 months. There were no severe AEs identified that were attributable to SP or SLD PQ administered during the 2018 pilot campaign. However, a similar MDA campaign in 2020 identified four cases of SCARs, which raises concerns about using SP for MDA during the current pandemic.Bill & Melinda Gates Foundation (OPP1114297) to the CDC Foundation as part of the Malaria Zero Consortium.Funding was provided by the U.S. Centers for Disease Control and Prevention and by a grant from the


This study aims to investigate the applicability of lower lid margin thickness (LLMT) measurements in adults with and without meibomian gland dysfunction (MGD) by optical coherence tomography (OCT) and keratograph.This is a cross-sectional, observational study. A hundred and eight volunteers aged 20 to 79, including 68 MGD patients and 40 normal subjects, were recruited. Using OCT and keratograph to measure the LLMT from the posterior lash line to anterior edge or outer edge of the tear meniscus was separately performed two times by the same person.P < 0.05), and intraclass correlation coefficient (ICC) demonstrated a good agreement in the LLMT measurements between two devices . Additionally, the LLMT in MGD patients was appeared to be positively correlated with meiboscore .The mean age of normal and MGD subjects was 50.5 ± 14.2 years and 55.8 ± 15.5 years, respectively. The LLMT with OCT and keratograph in MGD patients was significantly greater than that in normal subjects . In both normal and MGD subjects, the tear meniscus height and LLMT with OCT were both greater than that with keratograph (The OCT and keratograph were two reliable tools in the LLMT measurements, which may have potential applications for diagnosis and evaluation of MGD. Furthermore, we found that the LLMT measured by OCT was greater than that measured by keratograph. Meibomian gland dysfunction (MGD) is a clinically common ocular surface disease, which is closely related to evaporative dry eye . A cliniPopulation-based studies around the world have reported a prevalence of DED, with a range from approximately 5 to 50% . The teaIt is well recognized that rounding and thickening of the lid margin are a common feature of MGD , 24, whiAlthough several studies have assessed the agreement of TMH measurement between OCT and keratograph , 21, theThis was a single-center, prospective, cross-sectional, observational study, which was an extension of our previous serial studies –29. In tThe diagnosis of MGD was established if a patient met the following criteria , 30: (1)The study exclusion criteria were –29 oculaThe fluorescein tear film break-up time (FBUT) was measured using sterile sodium fluorescein strips . FBUT was measured three times for each eye, and then the average was recorded , 30.After FBUT measurements, corneal fluorescein staining (CFS) was observed under the slit-lamp biomicroscope with a cobalt blue filter. The cornea was split into 4 quadrants , and each quadrant was graded on a scale of 0 to 3: 0, no staining; 1, 1–30 dots; 2, > 30 dots without confluent patches; and 3, confluent patches, and/or filaments, and/or ulcer , 30.After CFS, the Schirmer test without anesthesia (SIT) was observed with a sterile Schirmer strip (Tianjin Jinming New Technology Development Co Ltd), which was placed in the temporal one-third of the lower eyelid cul-de-sac of each eye for 5 min , 30.The meibum quality grade scale of each of 8 glands in the central area of lower eyelid was 0 to 3 (0–24 points): 0, clear; 1, cloudy; 2, cloudy with granular; and 3, thick, like toothpaste , 30.According to the number of 5 glands in the central third of lower eyelid from which the secretion could be expressed, the meibum expressibility was graded on a scale of 0 to 3: 0, 5 glands expressible; 1, 3 to 4 glands expressible; 2, 1 to 2 glands expressible; and 3, no glands expressible , 30.The meibomian gland dropout grade (meiboscore) was observed by Keratograph 5 M . The meiboscore of each eyelid was evaluated: 0 = normal; 1 = dropout ≤ 1/3; 2 = dropout > 1/3 and ≤ 2/3; and 3 = dropout > 2/3) .Referring to our previous published articles , 30, allIn this study, the LLMT was defined as the distance from the posterior lash line to the anterior edge of the tear meniscus at the central eyelid Fig.  26–29].–29.26–29The LLMT measurements in 30 normal healthy volunteers were, respectively, performed by two well-trained operators (D.-H. W and X.-Q. L) with Spectralis OCT  and Keratograph 5 M  under the same conditions, and the protocol process was basically the same as the measurement of TMH –21. OperThe lower lid margin thicknesses of 108 subjects  were measured by the same examiner with OCT and keratograph. The protocol processes were basically the same as our previous researches –28. In bIt should be emphasized that because of the rounded contour of lid margin and the poor tissue penetration of OCT and keragraph devices, it was difficult to measure the full thickness of lid margin including the mucosa. We all knew that the palpebral border of lid margin corresponded with the MCJ, i.e., the position of anterior edge of the tear meniscus. Since the boundary of MCJ was not very clear in the images, we regarded the anterior edge of tear meniscus to replace the MCJ as the posterior boundary of the keratinized lid margin skin in this study. In addition, the LLMT in the OCT and keragraph images was the line length rather than the actual arc length. At present, it was almost impossible to directly measure the arc length in the images. Therefore, in order to get an accurate measurement, we used two devices to measure the same area of lower lid margin from the posterior lash line to the anterior edge of tear meniscus, which had been proved to be a relatively constant feature of the lid margin , 28.t test, nonparametric test, intraclass correlation coefficient (ICC), Bland–Altman plots, and Spearman’s rank correlation coefficient analysis. Receiver operating characteristic (ROC) curves with calculations of the area under the curve (AUC) were used to compare the diagnostic ability of LLMT measurements performed by OCT and keratograph for MGD. The total meiboscore of the upper and lower eyelids of both eyes was analyzed. P < 0.05 was considered significant. All analyses were performed with PASW Statistics version 25.0 .All data were presented as the mean ± standard deviations (SD). The statistical analyses were performed with Student’s P =  < 0.001 and < 0.001, respectively) . The LLMT measured by two operators with OCT and keratograph were 0.85 ± 0.16 mm, 0.86 ± 0.18 mm, 0.83 ± 0.17 mm, and 0.84 ± 0.17 mm, respectively. Bland–Altman plots and ICC demonstrated a good agreement in the LLMT measurements with OCT and keratograph between two operators  and 68 MGD subjects  was 50.5 ± 14.2 years and 55.8 ± 15.5 years, respectively. The detailed demographic characteristics and clinical parameters of the study are presented in Tables P < 0.05), and ICC and Bland–Altman plots demonstrated good agreement in the LLMT measurements between two devices    compared with normal subjects . However, the LLMT measured by OCT and keratograph in MGD patients was significantly greater than that in normal subjects . In both normal and MGD subjects, the TMH and LLMT measured by OCT were both greater than that measured by keratograph (02) Fig. .Fig. 3CoP = 0.45; 56.0 ± 17.4 vs. 55.2 ± 14.9 years, P = 0.56), and the LLMT measured by OCT and keratograph in males was about 0.1 mm thicker than that in females, but there seemed to be no significant difference . Additionally, the Spearman rank correlation coefficient demonstrated that the LLMT of both normal and MGD subjects measured by OCT and keratograph was significantly positively correlated with age .In both normal and MGD subjects, the average age between the sexes was not significantly different  was significantly greater than that in normal subjects (0.90 ± 0.20 and 0.86 ± 0.16 mm), which was basically consistent with our previous findings , and furIn addition, the AUC of the LLMT with OCT and keratograph is about 0.70, which suggested an approximately 70% chance that the ophthalmologists will correctly distinguish normal people from MGD patients through the LLMT measured by two devices. Since the lid margin changes in MGD were multifactorial, such as telangiectasia, dimpling or notching, irregularity, cicatrices, abnormal orifices, dilation of the meibomian gland ducts, and displacement of MCJ, in this study, to facilitate the LLMT measurements, we excluded MGD patients with irregular lid margin structures and did not investigate other factors, which might diminish the diagnostic efficacy of LLMT in distinguishing MGD subjects from healthy subjects.rs = 0.76 and 0.61, P =  < 0.001 and < 0.001, respectively), which was consistent with our previous observations [In MGD subjects, we found that the LLMT had an approximately linear relationship with meiboscore, which further supported the observations that the lid margin abnormalities were associated with meibomian gland dropout , 37. In rvations –29. Howervations  suggesteGenerally, a good agreement between two devices indicated that the LLMT measurements were reproducible. It will be of great value to investigate the LLMT with OCT and keratograph to discriminate MGD patients from normal subjects. In this study, since we excluded MGD patients with irregular lid margin structures, which might diminish the relevance of LLMT in MGD with age. In addition, exploring the relationship between the LLMT and the prognosis or severity of MGD will be the focus of our future work.However, several limitations of our study should be noted. (1) This study only measured the keratinized lid margin surface width excluding mucosa, not full length of the LMT. (2) In view of the slope of upper lid margin and the presence of upper eyelashes, and the optical properties of OCT and keratograph devices, we only measured the LLMT and did not measure the upper LMT. (3) In addition, we excluded MGD patients with the irregular lid margin structures and did not compare the difference of LLMT between early-stage and advanced MGD. Further studies were warranted to provide more information.The OCT and keratograph were two rapid, noninvasive methods for assessing the LLMT with acceptable repeatability. The LLMT in MGD patients was greater than that in normal subjects and mildly positively correlated with meiboscore, which indicated that the LLMT measurements might have potential applications for the diagnosis and evaluation of MGD. Furthermore, we found that the LLMT measured by OCT was greater than that measured by keratograph.


In malaria-endemic areas, children presenting to hospitals with a decreased level of consciousness remain a diagnostic dilemma. The definition of cerebral malaria in a comatose child demands exclusion of other possible reasons, which requires in-depth investigations that are not easily available. The aim of this study was to investigate the frequency and clinical characteristics of PCR-confirmed malaria in a cohort of children with a decreased level of consciousness, look for potential features that would aid in differentiating children with malaria from those without, and assess the performance of traditional thick film microscopy against the cytb-qPCR-method.A total of 345 children aged 30 days–15 years old, presenting to Hospital Pediátrico David Bernardino in Luanda, Angola, with a decreased level of consciousness  were prospectively enrolled during 2014–2017. Malaria was defined as a positive cytb-qPCR result on any occasion in hospital. The clinical course and laboratory parameters were compared between children with malaria and those without. The performance of thick film microscopy was analysed against the PCR method.Plasmodium falciparum species, and 82.6% (133/161) fulfilled the WHO criteria for severe malaria. Overall, children with malaria presented to hospital with a shorter duration of symptoms and less convulsions pre-admission compared to those without malaria. The median GCS score on admission was 8, which did not differ between children with or without malaria. Clinical findings on admission were mostly similar across the whole cohort, but an infection focus outside the central nervous system was more common in malaria-negative children. Moreover, severe anaemia, thrombocytopenia, and high CRP levels occurred more frequently in children with malaria. The case fatality ratio was 28.5% (91/319) and did not differ between parasitaemic children and those without malaria, although parasitaemic children died sooner after hospital admission. When neurological sequelae were also considered, a positive malaria test was associated with a better outcome. The performance of thick film microscopy against PCR yielded a sensitivity of 96.8% and a specificity of 82.7%.161 of 345 children (46.7%) had a positive malaria PCR test result. All cases were In this cohort of children with a decreased consciousness, the frequent presence of a malarial infection could not be judged from the clinical findings on admission, but the combination of profound aneamia, thrombocytopenia, and a high CRP level increased the odds of a positive malaria test result. Mortality remained high regardless of etiology, but malaria infection associated with fewer neurological deficits at discharge. Thick film microscopy performed well compared to the cytb-qPCR method.The online version contains supplementary material available at 10.1186/s12936-023-04556-9. Malaria accounted globally for more than 600,000 deaths in 2021, the majority of which were in children .In general, children with malaria were older and attended school or day-care more frequently than those without parasitaemia Table . MoreoveChildren with a positive malaria PCR result presented to hospital with a shorter duration of illness (median duration of four vs. five days). In addition, a higher frequency of children without malaria suffered from convulsions before hospital admission, compared to those with documented malaria Table .At the emergency department, signs of malnutrition or an infection outside the central nervous system were more common in malaria-negative patients Table . OverallIn terms of the CSF findings, most of the children showed no pleocytosis. The CSF protein level was slightly elevated in both groups, although higher in malaria-negative cases. Moreover, the CSF glucose level was lower in children without malaria Table . In totaIn general, children with malaria received more blood transfusions and malaria treatment, but less antibiotics, anticonvulsants, corticosteroids, acyclovir, or tuberculosis treatment Table . HoweverIn total, 28.5% 91/319) of the children died. The case fatality ratio did not significantly differ between those with malaria and those without , but children with malaria died closer to hospital admission . Using the PCR as the reference method, microscopy yielded a sensitivity of 96.8% (95% CI 90.1–99.3) and a specificity of 82.7% (95% CI 72.7–90.2). This corresponded to a positive predictive value of 86.5% (95% CI 80.0–91.2) and a negative predictive value of 95.7% (95% CI 88.0–98.6)  of the children with a positive microscopy result were PCR-negative, and 3/93 (3.2%) of the children with a negative microscopy result had malaria parasites in the blood detected by PCR. Within the PCR-negative group, children with a positive microscopy differed in terms of a lower haemoglobin level , while no significant differences were noted in other laboratory parameters on admission or the survival. As for the three PCR-positive children with a negative microscopy, one had culture-confirmed pneumococcal meningitis, but no differences in the blood test results emerged. Two of these children died, resulting in a higher case fatality ratio than in children with both positive microscopy and positive PCR test result (p = 0.04).In this cohort of children presenting to hospital with a decreased level of consciousness, half  had PCR-confirmed malaria parasitaemia. Few clinical findings on admission differentiated children with malaria from those without, although differences in laboratory findings clearly emerged. The case fatality ratio did not significantly differ between parasitaemic and non-parasitaemic children.Children with malaria were slightly older than those without malaria and attended school or day-care more often Table . OverallIn terms of laboratory parameters, children with malaria were more frequently anaemic and thrombocytopenic on admission and showed higher serum levels of CRP compared to those without parasitaemia Table . In lineChildren with malaria died earlier after hospital admission than children without. Indeed, death due to severe malaria typically occurs soon after hospital admission, the majority within 24 h , 22–24. On the other hand, the incidence of neurological sequelae Table  remainedThe given treatment—which often included both anti-malarials and antibiotics—is in line with the WHO guideline for treatment of severe malaria : In endeIt remains unclear how many of these children with malaria had an impaired consciousness due to severe malaria. Of the 161 children with a positive malaria PCR result, only two had culture-confirmed bacterial meningitis. However, 17% of them received antibiotic treatment prior to hospital admission. Profound anaemia and thrombocytopenia were more common in malaria-positive children, which might point towards the parasitaemia being significant.There is also uncertainty about the number of children with CM, as severe anaemia or acidosis, hypoglycaemia, post-ictal status or malaria-associated acute kidney injury and subsequent uraemia may also impair consciousness. Among the 161 children with malaria parasitaemia, 42 had other identified possible causes of decreased consciousness , leaving 119 children with strictly defined CM. Nonetheless, as different signs of severe falciparum malaria commonly co-existed, identification of one sign does obviously not exclude another . UnfortuThe performance of thick film microscopy against PCR yielded a sensitivity of 96.8% and a specificity of 82.7%. Compared to previous reports, which have demonstrated a highly variable performance, thick film microscopy at Hospital Pediátrico David Bernardino in Luanda seemed to perform reasonably well , 29, 30.The findings of this study must be interpreted in light of potential limitations. The sample for the malaria PCR analysis was taken on the third day or later in 13% of children, and the exact date of sampling was unknown in 21%. Although not probable, it cannot be excluded that some of the children with parasitaemia acquired the infection in hospital, which would lead to an overestimation of the baseline incidence of malaria. It is likewise possible that some of the negative samples taken later during the treatment were positive on admission, leading to misclassification of these patients.Some data were also missing for the definition of severe malaria according to the WHO, such as serum bilirubin levels and severity of acidosis. These data would have been of interest, especially while addressing the proportion of CM. Moreover, also information on the treatment given before hospital admission was limited in some patients. Finally, the true incidence of decreased consciousness due to severe malaria (including CM) in this cohort remains uncertain.In Luanda, approximately half of the children presenting to hospital with a decreased level of consciousness have malaria parasitaemia. Few clinical characteristics aid in differentiating children with parasitaemia from those without, but blood count and CRP analyses might be helpful in assessing the risk of malaria infection. Targeted thick film microscopy on admission remains an adequate tool for diagnosing malaria in this population.Despite that most of these severely ill children received both antibiotic and antimalarial treatment, 28.5% still died. Although the case fatality ratio did not significantly differ between parasitaemic children and those without parasitaemia, a positive malaria PCR result was associated with a better prognosis overall. Nonetheless, regardless of etiology, children presenting to hospital with an impaired consciousness in these circumstances require meticulous attention in order to optimize the given treatment and improve the outcome.Additional file 1: Table S1. Performance of microscopy against the cytb-qPCR method. Description: A table showing the positive and negative results of the cytb-qPCR method and thick film microscopy from samples obtained on admission.


P. falciparum infection and underlying host adaptive immune responses in an area with a high prevalence of asymptomatic parasitaemia in Cameroon. A cross-sectional study of 353 individuals aged 2 to 86 years (median age = 16 years) was conducted in five villages in the Centre Region of Cameroon. Plasmodium falciparum infection was detected by multiplex nested PCR in 316 samples, of which 278 were successfully genotyped. Of these, 60.1% (167/278) were polyclonal infections, the majority (80.2%) of which were from asymptomatic carriers. Host-parasite factors associated with polyclonal infection in the study population included peripheral blood parasite density, participant age and village of residence. The number of parasite clones per infected sample increased significantly with parasite density  but decreased with participant age . Parasitaemia and the number of clones per sample correlated negatively with total plasma levels of IgG antibodies to three highly reactive P. falciparum antigens  and two soluble antigen extracts (merozoite and mixed stage antigens). Surprisingly, we observed no association between the frequency of polyclonal infection and susceptibility to clinical disease as assessed by the recent occurrence of malarial symptoms or duration since the previous fever episode. Overall, the data indicate that in areas with the high perennial transmission of P. falciparum, parasite polyclonality is dependent on underlying host antibody responses, with the majority of polyclonal infections occurring in persons with low levels of protective anti-plasmodial antibodies.Malaria remains a major public health problem worldwide, with eradication efforts thwarted by drug and insecticide resistance and the lack of a broadly effective malaria vaccine. In continuously exposed communities, polyclonal infections are thought to reduce the risk of severe disease and promote the establishment of asymptomatic infections. We sought to investigate the relationship between the complexity of  P. falciparum, infection with multiple clones of the parasite is common, as well as a high prevalence of asymptomatic parasitaemia [Malaria remains a major public health threat, with morbidity and mortality rates on the rise in several parts of the world, as revealed in the latest editions of the World Malaria Report ,2,3. Effsitaemia . WhetherP. falciparum infection in an area of high perennial transmission and high prevalence of asymptomatic malaria parasitaemia in Cameroon, one of 11 “high burden high impact” countries that accounted for 68% of the global estimated malaria cases and 70% of global estimated deaths in 2021 [P. falciparum [Plasmodium parasites in Cameroon is very limited, and none of the published work explored the host and parasite factors associated with polyclonal infection. We found a strong positive correlation between a multiplicity of infection and parasite density and that the frequency of polyclonal infection was highest in children and generally in participants with low levels of IgG antibodies against protective P. falciparum antigens, suggesting the dependence of both parasite density and polyclonality on the underlying host adaptive immune responses.In this study, we sought to assess the prevalence and the host and parasite factors associated with multiclonal  in 2021 . The coulciparum . Data onThe study was approved by the National Ethics Committee for Human Health Research of Cameroon  and authorized by the Ministry of Public Health (D30-989/L/MINSANTE/SG/DROS). The field studies were also approved by local administrative authorities, including the senior divisional officer in Esse and village chiefs. Informed consent was obtained from participants over 19 years of age and from parents or legal representatives of children under the age of 19. Assent was also obtained from all children aged 12 to 19 years.This study was conducted in five villages (Afanetouana (Af), Koutou (Ko), Meboe (Me), Ondoundou (On), and Ntouessong (Nt)) in the locality of Esse, Central Region of Cameroon, and belonging to the Yembouni population group . The stuPlasmodium species were performed by multiplex nested PCR and light microscopy, respectively [The detection and quantification of infecting ectively . BrieflyPlasmodium species infections by multiplex nested PCR targeting the 18S ssrRNA genes of P. falciparum, P. ovale, P. malariae, and P. vivax, as described by Snounou et al. [Plasmodium genus-specific outer primers and 10 µL of Platinum green Hot start PCR 2X master mix . Nested PCR was performed with 2 μL of the PCR product, 0.2 µM species-specific primers and 10 µL of Platinum green Hot start PCR 2X master mix. Both PCR steps were performed under the same cycling conditions, which included initial denaturation and enzyme activation at 95 °C for 15 min, 43 cycles of 95 °C for 45 s and 55 °C for 90 s, and a final extension at 72 °C for 5 min . Asymptomatic infections were defined as the presence of Plasmodium parasites in peripheral blood, detected by multiplex nested PCR, in the absence of fever (temperature < 37.5 °C) at least 48 h before enrollment into the study. Symptomatic infections were defined as positive for Plasmodium infection by any one of three methods  and a history of fever (temperature ≥ 37.5 °C) within 48 h before enrollment. Participants with negative results for all three tests were considered uninfected with malaria parasites.Genomic DNA was extracted from dried blood spots (Whatman grade 3) containing 50 µL of whole blood using the Chelex method  and usedu et al. . For theor 5 min . PCR assP. falciparum genotyping was performed targeting two well-characterized polymorphic markers, msp2 and polyα, as previously described [escribed ,33,34,35msp2 genotype was identified based on the size of the PCR products using a 2% agarose gel containing 0.01% of ethidium bromide and analyzed under a UV transilluminator. Identification of the polyα polymorphism was performed after SDS-PAGE on a 12% polyacrylamide gel. The amplification product was identified after staining the polyacrylamide gel with 0.01% ethidium bromide on a shaker at room temperature for 30 min and analyzed under a UV transilluminator. Expected heterozygosity (He), which is a key determinant of population structure, representing the probability of being infected by two parasite populations with different alleles at a given locus, was determined according to the formula He = n/(n − 1) × . Samples with more than one allele (MOI > 1) were classified as polyclonal infections, whereas those with a single allele by both genotyping methods were considered monoclonal infections.All reactions were carried out in a final volume of 20 µL containing 200 nM of each primer, 4 µL of 5× Hot FIREpol blend master mix , and nuclease-free water. In the first PCR, 5 µL of purified DNA sample was used, and in the nested reaction, 2 µL of amplicon was used. The iversity ,36. CompPf) and merozoite antigen (anti-MZ) of P. falciparum were measured by indirect ELISA. The soluble extracts were obtained from laboratory-maintained P. falciparum 3D7 parasites by freeze-thaw fractionation in PBS, pH 7.4 containing protease inhibitors, whereas the P. falciparum Merozoite Surface Proteins were produced using a baculovirus insect cell expression system (MSP-1p19 and MSP-4p20) or in E. coli (MSP3) [Levels of plasma IgG to four highly reactive recombinant antigens: MSP-1p19, MSP3, MSP4p20, and EBA175 (MRA-1162) ,38, and i (MSP3) ,39,40. Mp < 0.05 were included in the multivariate analysis. The Mann–Whitney test was used to compare the median between the two groups. Spearman’s test was used to determine the correlation between two quantitative parameters. Participants were classified as anemic based on WHO classification guidelines using hemoglobin levels for each age group and gender [All statistical analyses were performed using Stata/MP 13.0 and GraphPad Prism 8.0.1, Harvey Motulsky, San Diego, CA, USA). Univariate and multivariate logistic regression models were used to determine the host and parasite factors associated with polyclonal infections in the study area. Only associated variables with d gender . All parP. falciparum infection, of which 88.6% were Pf mono-infections, 8.9% were Pf + Pm, 1.9% were Pf + Po, and 0.6% were Pf + Pm + Po. The demographic, clinical and parasitological characteristics of the study population are described elsewhere [Pf-positive samples resulted in 250 (79.1%) positive amplifications at the msp2 locus and 215 (68.0%) at the polyα locus, for a combined genotyping success rate of 88.0% (278/316). Of the 38 PCR-positive but non-genotyped samples, 23 (60.5%) were submicroscopic infections and 15 (39.5%) were low-density infections . The characteristics of the genotyped and non-genotyped populations are presented in Of 353 individuals tested for malaria parasites by multiplex nested PCR, 316 (89.5%) were positive for lsewhere . Genotypmsp2 locus, whereas 21 alleles were identified at the polyα locus at frequencies between 1.4% and 20.5%.As shown in msp2 genotyping and 1.67 (range: 1–6 clones) by polyα microsatellite analyses, for a combined MOI of 1.95 in the study population. The mean MOI was 1.58 (range: 1–5 clones) in individuals with submicroscopic infection and 2.1 (range: 1–8 clones) in the microscopy-positive subjects (p < 0.0001). It was 1.91 (range: 1–6 clones) in the asymptomatic individuals compared to 2.08 (range: 1–8 clones) in the symptomatic population (p = 0.6931), and it was 2.3 (range: 1–8 clones) in children compared to 1.60 (range: 1–5) in adults (p < 0.0001). The mean number of clones per infected subject varied according to locality, with Ntouessong having the highest mean MOI , followed by Afanetouana , Ondoundou , Meboe  and Koutou . Expected heterozygosity (He) was generally high  in all the villages studied. The mean expected heterozygosity was 0.82 considering the msp2 alleles and 0.85 when considering the polyα microsatellite marker. These results indicate a high genetic diversity of malaria parasites in the study population, with children and microscopically positive participants carrying the highest number of parasite clones.The mean number of parasite clones per infected participant (MOI) was 1.70 (range: 1–8 clones) by p < 0.0001). It was 60.36% in asymptomatic participants as against 58.93% in the febrile subjects (p = 0.8450). Approximately 63.47% (106/167) of individuals with polyclonal infections had parasitaemia greater than the population median, compared to only 28.82% (32/111) of high parasitaemia among individuals with monoclonal infections. The frequency of polyclonal infection varied with age and the participant’s village. It was 88.24% in children under five years old, 76.56% in children between 5–10 years old, 66.67% in 10–15 years old, and 46.85% in children and adults greater than or equal to 15 years of age. The frequency of polyclonal infection was highest in Ntouessong (74.3%), followed by Afanetouana (65.0%), Ondoundou (63.8%), Meboe (49.4%) and Koutou (47.7%). Together, these data suggest a high frequency of polyclonal P. falciparum infection in the study area and its probable association with age, parasite density and participant’s village.The frequency of polyclonal infection in the study population, taking into account both allelic markers, was 60.1% (167/278), of which 80.2% were asymptomatic infections. The frequency was 37.21% in submicroscopic and 70.31% in microscopy-positive individuals  and were four times more likely to occur in participants with high parasite densities than those with low parasitaemia . Median parasite densities were generally higher in participants with polyclonal infections than those with monoclonal infections, and the number of parasite clones increased significantly with parasite density    . In contctively) . These fp = 0.5082), suggesting a minor role of host exposure in the acquisition of polyclonal infection. Additionally, polyclonal infections were not associated with clinical status (asymptomatic or symptomatic) or with time to previous fever episode  and two antigen extracts (mixed stage extract and merozoite extract) were assessed by indirect ELISA. As shown in p = 0.0005; MSP3: r = −0.0064, p = 0.9334; EBA175: r = −0.0269, p = 0.7243; anti-Pf: r = −0.2994, p < 0.0001; and anti-MZ: r = −0.1564, p = 0.0393), but correlated positively with the IgG level to the recombinant MSP4p20 protein .To investigate the impact of underlying host antibody responses on the complexity of infection and frequency of polyclonal infection in the area, plasma IgG to four highly reactive p = 0.0177; MSP3: r = −0.1130, p = 0.2171; EBA175: r = −0.2161, p = 0.0173; anti-Pf: r = −0.4692, p < 0.0001; and anti-MZ: r = −0.2810, p = 0.0018), whereas a positive correlation was observed for IgG levels to MSP4p20 . Plasma IgG levels increased with age for all five antigens , and decreased for the MSP4p20 antigen . Overall, these data suggest a potential role for underlying host antibody responses in limiting blood parasite densities as well as the number of infecting parasite clones per individual, with the highest parasitemia and number of clones observed generally in participants with low plasma levels of anti-Plasmodium IgG.Similarly, a negative correlation was observed between parasite density and IgG levels against the five antigens  were amplified by nested PCR, and samples were considered polyclonal if multiple alleles were detected by either one or both genotyping methods. Association of the underlying host adaptive immune responses with polyclonality (frequency and MOI) was assessed by measuring total IgG levels against several highly reactive P. falciparum antigens and against total soluble extracts (merozoite and mixed-stage) of a laboratory strain .Studies undertaken in malaria-endemic areas worldwide, particularly in areas of perennial  attacks ,19,20. TConsistent with published data on the genetic diversity of malaria parasites in hyperendemic areas ,44,45,46P. falciparum, as well as against the merozoite soluble extract. Indeed, our study is the first to report an association between the complexity of P. falciparum infection and anti-Plasmodium antibody responses. In a previous study in Nigeria [PfCSP, a circumsporozoite protein and marker of exposure to P. falciparum, despite a strong correlation with age. In general, with the exception of IgG levels against the MSP4p20 antigen, IgG levels increased with age, consistent with the acquisition of antimalarial immunity in long-term residents of malaria endemic zones [P. falciparum infection [Plasmodium antibodies are generally clonotype-specific and may be short-lived [Parasitaemia and clone number correlated negatively with IgG levels against multiple highly reactive and protective antigens of  Nigeria , the autic zones ,12. The nfection ,49. Overrt-lived ,15,38. TIn conclusion, the findings reported in this work are consistent with a dependence, in areas of high parasite diversity, of both parasitaemia and clonality on the degree of protective antibody immunity at the time of infection. A major drawback in this study concerns the limited sample size relative to the total population of the study area and our failure to genotype up to 12% of the PCR-confirmed infections, which may have skewed some of the factors studied. Further study of the parasite determinants of multiclonal host immunity could lead to the discovery of more effective vaccines to accelerate malaria elimination efforts globally.


Malaria remains the leading cause of mortality and morbidity in young children in sub-Saharan Africa. To prevent malaria in children living in moderate-to-high malaria transmission areas, the World Health Organization has recommended perennial malaria chemoprevention (PMC). Prior to piloting PMC implementation in southern Togo, a household survey was conducted to estimate malaria infection prevalence in children under 2 years of age (U2).Plasmodium infection, defined as a positive rapid diagnostic test (RDT), was estimated with 95% confidence interval (CI). Clinical malaria was defined as having a positive RDT plus fever (≥ 37.5 °C) or history of fever in the last 24 h. Mixed-effects logistic regression models were used to assess the child’s, caretaker’s, and household’s factors associated with malaria infection.A cross-sectional community-based household survey was conducted in the Haho district in the Togo Plateaux region. A three-stage random sampling method was used to select study participants aged 10–23 months whose caretakers gave informed consent. The prevalence of A total of 685 children were included in the survey conducted January–February in 2022 (dry season). Median age was 17 months (interquartile range: 13–21). About 80% of the children slept under a bed net the night before the interview. Malaria infection prevalence was 32.1% (95% CI 27.7–37.0) with significant area variation (cluster range: 0.0–73.3). Prevalence of clinical malaria was 15.4% (95% CI 12.2–19.2). Children whose caretakers were animist  and those living in mother-headed households  were more likely to have a positive RDT. Living more than 5 km away from the nearest health facility  and presence of two or more under-5-years children in the household  were also associated with increased risk of infection.One-third of the children U2 who participated in this survey had malaria infection, thus PMC could be a promising strategy to reduce malaria burden in young children in Plateaux region. Reinforcement of outreach services and targeting the poorest households should be prioritized to reduce the inequity in malaria prevention in children exposed to the infection. In recent decades, progress has been made to reduce malaria burden in high endemic areas. However, malaria remains a serious public health problem in Togo affecting a large number of people . In 2019Plasmodium infection and associated risk factors in children under 2 years of age (U2), who are the target population of PMC.In 2010, in the areas where malaria transmission is perennial, the World Health Organization (WHO) recommended intermittent preventive treatment of malaria in infants (IPTi) to those living in moderate-to-high malaria transmission areas where parasitic resistance to sulfadoxine-pyrimethamine (SP) is low . IPTi inA cross-sectional community-based household survey was conducted in the Haho district from January to February 2022. The Haho district is located in the Plateaux region approximately 100 km from the capital city Lomé. Malaria transmission in the area occurs along the year with peaks in two rainy seasons (March–July and September–October) . TherefoThe survey included children U2 who live in the district and meet the following criteria: (i) age 10–23 months (inclusive), and (ii) the child’s caretaker’s agreement to participate in the research by signing the informed consent form. To calculate the sample size, a design effect of 2, 5% precision and 10% non-response as well as the malaria prevalence estimate in Togo in 2017 (28%) were assumed . Based oA standardized questionnaire was administered through the REDCap mobile application to collect socio-demographic and behavioural data on the children , caretakers and household heads  as well as caretakers’ knowledge and perceptions about malaria . InformaData collection was carried out by 25 teams of two health professionals who received a 4 day training session that covered research protocol, informed consent procedures, questionnaire administration using tablets, household and child selection, and the use of RDTs. Each team was assisted by a community health worker (CHW) who facilitated access to the households’ members.To summarize quantitative and qualitative data, the median with interquartile range (IQR) and proportion with a 95% confidence interval (CI) were used respectively. Data were weighted to reflect the multi-stage cluster sampling design. Weights were calculated for each sampling stage while those in the third stage, namely the random selection of a child, were applied only to the data from the households having multiple eligible children. The prevalence of malaria infection was defined as a weighted proportion of children who was tested positive for malaria RDT among all children. Similarly, the prevalence of clinical malaria among all participating children was estimated as a weighted proportion of children with a positive RDT and history of fever (≥ 37.5 °C) within the past 24 h or on the day of the survey.To assess factors associated with children’s malaria infection while accounting for correlation between clusters, mixed-effect logistic regression models were built with clusters included as a random effect. Factors tested in the models were selected a priori based on previous knowledge about predictors of malaria in children and adults –26. ChilFirst, the association between malaria infection and children’s characteristics including age, use of a mosquito net the night before the interview, immunization status, and vitamin A uptake was tested. Children’s immunization status was classified into three categories  according to the information available in the child’s EPI card or on the basis of the caretaker’s self-declaration if the card could not be located at the time of the interview. Fully-immunized children were those who have received all the routine scheduled vaccines by 9 months of age according to Togo’s national EPI schedule. If any of the vaccines were missed, children were considered as partially immunized. Vitamin A was recommended at the age of 9 months in Togo, thus vitamin A uptake was defined as binary .Second, the association between the child’s malaria infection and the characteristics of caretakers or household heads was investigated. In this model, the factors significantly associated in the first model (P < 0.05) as well as caretakers’ characteristics including age, marital status, educational level , literacy , ethnic group, religion , income source , and possession of child’s EPI card  were included. The literacy level was defined by reading and writing exercises included in the questionnaire. Caretakers who were able to read and write the requested sentences without any difficulty were classified as “fully literate.” Those with difficulties in at least one of those exercises were defined as “partially literate,” and anyone incapable of reading or writing was considered as “illiterate.” In this model, the variables related to the characteristics of household heads such as his/her relationship with the child , education level, and income source, were also included.Finally, a model was built with all the variables significantly associated with the outcome in the previous model (P < 0.05) and the households’ characteristics including the number of children U5 (one vs. two or more) and the village’s location in terms of the distance from the nearest health facility (< 5 vs. ≥ 5 km).P-value < 0.05 was considered as statistically significant in the final model. Statistical analyses were performed using the lme4 package in R version 3.6.2  .Based on the enumeration exercise, 12,506 households were identified in the 57 clusters. A total of 3518 households were visited and of 685 children were included in the survey  had a median age of 17 months (IQR: 13–21) (Table % girls hCaretakers were mostly mothers (93.5%) and had a median age of 27 years (IQR: 23–33). Almost all declared being married or in a relationship (95.4%). The majority were self-employed (89.0%). They had low education levels , and few were fully literate (21.2%). Around 60% were Christians, and 31.0% were animists. Only 58.3% declared being in possession of the child’s EPI card.In 77.1% of the households, the household head was the child’s father. Only 26.8% of the household heads had secondary or higher education, and the majority (87.9%) were self-employed. In more than 60% of the households, the child participant was the only U5 child.The caretakers had a good knowledge about malaria (94.7%), its cause, and how to prevent it Table . More thThe prevalence of malaria infection was 32.1% (95% CI 27.7–37.0), with significant variation across areas (cluster range: 0.0–73.3) . Village location was found to be a significant predictor of children’s infection status. When the village was located more than 5 km away from the nearest health facility, the children were more likely to have a positive RDT . Also, children living in a household where there were two or more children U5 were at a significantly higher risk of infection . Association with the caretaker’s religion and the type of household head remained significant in the final model , whereas children’s age showed no association .Plasmodium infection was 32.1% among children in the survey with significant heterogeneity across areas. One-half of them had clinical malaria (15.4%). High prevalence was observed in certain parts of the district. This study is the first to report such detailed sub-district-level data on malaria prevalence in this area.The prevalence of The 2020 national survey reported a prevalence of 48.7% in the Plateaux region, measured in November and December by microscopy . While tIn the present study, several factors were associated with a child’s infection status, in line with the findings from previous studies. The body of evidence has shown that low socioeconomic status was a significant predictor of malaria –26, 31. Children living in a cluster located more than 5 km away from a health facility were at increased risk of malaria infection. In Togo, in order to reach the population living in such remote areas, CHWs are deployed to relay essential malaria-related services including diagnosis and treatment of uncomplicated cases with artemisinin-based combination therapy. Routine EPI services are also delivered through outreach activities together with other mother-and-child health services including antenatal care, preventive treatment of malaria for pregnant women, and distribution of bed nets to pregnant women and children under 12 months of age. Further investigations of the areas where extremely high prevalence was observed  found that almost one-half were located more than 10 km away from the nearest health facility. Of these, half were a distance greater than 20 km. While such high prevalence can be explained by reduced access to healthcare and information, it might also be the consequences of environmental risk factors including land use for farming, presence of livestock, proximity to breeding sites, and housing quality, which could not be untangled due to lack of information. Nonetheless, the findings suggest that more concerted efforts are needed to address the vulnerability of children living in such hard-to-reach areas. The current efforts of the Togolese Ministry of Health to integrate CHWs more formally into the health system should thus be seen as a positive step towards improving health equity and reducing the burden of malaria in these high transmission areas.Anopheles gambiae sensu lato (s.l.), the major malaria vector in the country, particularly to dichlorodiphenyltrichloroethane and pyrethroids [Lack of association between a child’s infection outcome and bed net use has been reported previously , 31, 34.ethroids –37. Thisethroids . The finethroids . Despiteethroids .An alternative prevention strategy such as PMC can further strengthen existing efforts and contribute significantly to reducing malaria burden in the area. The caretakers’ high acceptability of preventive drugs, as demonstrated in the survey, is encouraging and will greatly facilitate the introduction of PMC. In Sierra Leone, the only country where malaria chemoprevention has been implemented as part of the national strategy in perennial transmission settings, children U2 who have received three doses of SP were less likely to have malaria infection  . The preThis study provided information on the local malaria epidemiology in the Haho district before the introduction of pilot PMC implementation and identified risk factors to guide more effective malaria control measures in the area. The malaria prevalence was 32% with significant area variation. Coupled with the existing preventive strategy, PMC can play an important role in reducing malaria burden in this high transmission setting. To better protect children in the second year of life, the NMCP might consider means other than the routine EPI platform to deliver PMC. Delivering an effective malaria preventive strategy should be viewed within a larger framework of interventions that aim to improve the socioeconomic status of poor households. Efforts to reach children living in some hard-to-reach areas should continue and be strengthened in order to reduce inequity in their health outcome.


Malaria is one of the most dangerous infectious diseases in the world. It occurs in tropical and subtropical regions and affects about 40% of the world´s population. In endemic regions, an estimated 200 million people contract malaria each year. Three-quarters of all global deaths (about 600 per year) are children under 5 years of age. Thus, malaria is one of the most relevant tropical and also childhood diseases in the world. Thanks to various public health measures such as vector control through mosquito nets or the targeted use of insecticides as well as the use of antimalarial prophylaxis drugs, the incidence has already been successfully reduced in recent years. However, to reduce the risk of malaria and to protect children effectively, further measures are necessary. An important part of these measures is an effective vaccination against malaria. However, the history of research shows that the development of an effective malaria vaccine is not an easy undertaking and is associated with some complications. Research into possible vaccines began as early as the 1960s. However, the results achieved were rather sobering and the various vaccines fell short of their expectations. It was not until 2015 that the vaccine RTS,S/AS01 received a positive evaluation from the European Medicines Agency. Since then, the vaccine has been tested in Africa. However, with the COVID-19 pandemic, there are new developments in vaccine research that could also benefit malaria research. These include, among others, the so-called mRNA vaccines. Already in the early 1990s, an immune response triggered by an mRNA vaccine was described for the first time. Since then, mRNA vaccines have been researched and discussed for possible prophylaxis. However, it was not until the COVID-19 pandemic that these vaccines experienced a veritable progress. mRNA vaccines against SARS-CoV-2 were rapidly developed and achieved high efficacy in studies. Based on this success, it is not surprising that companies are also focusing on other diseases and pathogens. Besides viral diseases, such as influenza or AIDS, malaria is high on this list. Many pharmaceutical companies (including the German companies BioNTech and CureVac) have already confirmed that they are researching mRNA vaccines against malaria. However, this is not an easy task. The aim of this article is to describe and discuss possible antigens that could be considered for mRNA vaccination. However, this topic is currently still very speculative. Plasmodium. Approximately 40% of the world's population live in areas, where malaria is endemic. It is estimated that approximately 200 million people become ill and 600,000 people die each year. Of these 600,000 deaths, three-quarters are children under the age of 5 . Furthermore, the disease is ancient and closely linked to human history [Plasmodium falciparum. The maps were derived from the Malaria Atlas Project, a Collaborating Center in Geospital Disease Modelling of the WHO. The Malaria Atlas Project was launched in 2005 [Malaria is one of the most relevant infectious diseases in the world . The dis history . As a tr in 2005 .Fig. 1ThPlasmodium. The genus is species-rich and infects reptiles, birds as well as mammals. Five species are relevant to humans: Plasmodium falciparum, Plasmodium vivax, Plasmodium ovale, Plasmodium malariae, and Plasmodium knowlesi. All Plasmodium species undergo host switching. They are transmitted by mosquitoes of the genus Anopheles, which are also important as definitive hosts. For this reason, the management of the Anopheles host are relevant public health measures important for the control of the disease. Plasmodium species can be found worldwide.The pathogens that cause malaria are unicellular parasites of the genus Anopheles consists of 240 species. Only 40 of these have been shown to transmit malaria [Anopheles vector, vector control plays a very important aspect in global malaria management [The genus of the vector  malaria , 11. The malaria –15. Sincnagement , 17. Onenagement –20. Nowanagement , 22.Plasmodium can be transmitted to that human. In humans, the sporozoites first migrate to the liver to infect hepatocytes. In these, they develop into tissue schizonts, which can be up to 80 µm in size. Tissue schizonts contain merozoites that are released into the blood after maturation. Because this initial phase occurs in liver cells, it is referred to as the liver stage, or the pre- or exoerythrocytic phase. This phase is typically asymptomatic. P. ovale and P. vivax are able to form so-called hypnozoites. These cells are a dormant form of plasmodia that linger in the hepatocytes (in some cases for up to years) and can later cause a so-called relapse. The signal that triggers this relapse is still unknown [Anopheles mosquito during the blood meal. Sexual reproduction now takes place in the mosquito . In the intestine of the mosquito, the two gametes fuse to form a zygote. This develops first into a motile ookinete (in the intestine) and finally into an oocyst . In this oocyst new sporozoites are formed, which migrate maturely from the intestine into the salivary glands and are transferred to another human at the next blood meal of the mosquito. Thus, the life cycle is complete. Sporozoite development in the mosquito can take up to 4 weeks, depending on the species and temperature. Neither disease nor symptoms occur in mosquitoes [When an infected Anopheles mosquito bites a human to suck blood, sporozoites of  unknown . The mersquitoes , 25.In the science of parasitology, the different life cycles play a crucial role. Malaria is no exception. Knowledge of life cycles allows the development of different interventions that attack different stages of the life cycle. The mosquito nets described above, which decrease sporozoite transmission by preventing a mosquito bite, are just one example. Chemical methods such as the development of new therapeutics and prophylactics can also be guided by the life cycle, for example, by targeting specific proteins or relevant cell structures. Vaccines are one such example. However, past experience shows that developing of a potent vaccine against malaria is difficult and challenging. In the context of the SARS-CoV-2 pandemic, a new class of vaccines emerged that can and very likely will benefit the development of other vaccines against other pathogens. We are talking about mRNA vaccines, which are based on a messenger RNA that introduces an antigen into the body and thus triggers an immune response. Although mRNA-based vaccines have been the subject of research for some time, the first vaccines were not approved until the 2020s in the context of the SARS-CoV-2 pandemic. The vaccines were developed by various companies, such as BioNTech in Germany, Pfizer or MODERNA, both from the USA. Due to the success of the COVID-19 vaccines, many companies have already announced plans to expand their research to other diseases. In addition to diseases, such as influenza or other coronavirus infections, malaria is high on the agenda here, probably also for marketing reasons. Companies such as BioNTech from Mainz or CureVac from Tübingen have already announced to work on mRNA vaccines against malaria , 27. CoumRNA was first described in 1961 by Sydney Brenner, François Jacob and Matthew Meselson as a transporter of information relevant to biosynthesis . This diin vitro was successfully introduced into cells. This time without the use of a transfection reagent [In 1971, there was another milestone relevant to the development of mRNA vaccines. In that year, scientists succeeded for the first time in introducing foreign RNA into frog cells. The introduced RNA was isolated from rabbit reticulocytes. The work demonstrated that cells  or the cell's own translational machinery can also work with RNA derived from other cell types and from completely different species . In 1978 reagent . Both fiThe first RNA-based vaccines were described in 1993 and 1994. In these years, a cellular immune response was elicited for the first time , 39. In Plasmodium [Plasmodium. However, in smaller and more private settings, such as indoor spraying, DDT is still recommended and used [Plasmodium to chloroquine. However, the efficacy and tolerability of this combination is generally considered to be negative. Nowadays, this combination is no longer commonly used [As described above, the development of a potent malaria vaccine is the goal of many companies and universities. However, the development is not easy and associated with obstacles and difficulties. The development of an effective malaria vaccine is by no means new. As early as the 1960s, malaria was thought to have been conquered. Targeted public health interventions and programs (including programs established during World War II) resulted in a significant drop in cases. However, these rose again significantly at the end of the 1960s. Today, these renewed increases are thought to be due to resistance to DDT and chloroquine developed by asmodium . DDT, shasmodium . The WHOand used , 51. Chlnly used , 53.To counter the problem of increasing resistance, vaccines have been increasingly discussed since the 1960s. Health professionals, but also politicians, hope for a potent malaria vaccine to control and reduce the number of cases of this disease. The wish of a complete eradication, as it was still discussed in the 60s, for example, is nowadays no longer asked by anyone and is considered very unlikely . That vaToday, several vaccine platforms are already available, which differ in their mechanisms of action. Three vaccine classes are distinguished: pre-erythrocyte vaccines, blood-phase vaccines, and transmission blocker vaccines. While pre-erythrocyte vaccines and blood-phase vaccines are primarily intended to elicit an immune response, transmission blocker vaccines are intended to prevent transmission to humans and thus the spread of disease . They acPlasmodium falciparum version 66, also referred to as SPf66, is among the first vaccines developed against malaria. It is based on chemically synthesized 45-amino acid peptides derived from fractions of four different proteins extracted from Plasmodium falciparum [P. falciparum was reduced by 28%. However, SPf66 showed no effect on P. vivax. The same was true for hospitalizations for severe malaria [Serum lciparum . The vaclciparum . Pilot t malaria . Further malaria , 64. Todtm [Plasmodium and located on its cell surface. In studies, RTS,S/AS01 achieved 30% efficacy against uncomplicated malaria [RTS,S/AS01 is one of the most promising vaccines currently available. Developed by the British company GlaxoSmithKline, RTS,S/AS01, which is marketed under the name Mosquirixtm . It targ malaria . As of A malaria , 67. As  malaria . However malaria , 69. In  malaria , 71. The malaria , 73. Dos malaria .In addition to RTS,S/AS01, another vaccine targets the CSP. R21/Matrix-M is a relatively new vaccine being developed by the University of Oxford, among others. In a phase II trial conducted in Burkina Faso, a total of 450 children aged 5–17 months were vaccinated. They received one dose three times at 4-week intervals during the malaria season. A rabies vaccine was used as a control. An efficacy of up to 77% was achieved in the study. This efficacy is higher than other vaccine candidates, whose efficacy is approximately 56% –77.Plasmodium falciparum, SPZ for sporozoite. PfSPZ has already shown good results in small studies [4 PfSPZ prevented infection in 9 of 9 volunteers. Ten weeks after the dose, the volunteers were infected with malaria in a controlled manner [4 PfSPZ achieved 100% protection, a dose of 3.2 × 103 achieved 67% protection, and a dose of 1.28 × 104 achieved 33% protection [PfSPZ is another candidate being developed by Rockville, Maryland-based Sanaria Inc. PfSPZ uses sporozoites that are unable to divide to trigger an immune response in the body. To prevent division, these sporozoites are irradiated with radioactive radiation to weaken them. The name of the vaccine is derived from this process: Pf stands for  studies , 79. ThrPlasmodium infections remains one of the best targets for live attenuated vaccines [Another possibility to develop a robust vaccine against malaria is the use of genetically modified plasmodia. For example, rodents "vaccinated" with uis3-deficient sporozoites showed a robust immune response to them . UIS3 , 40% (2-week pretreatment) and finally to 0% (3-week pretreatment) [in vivo and not in vitro. Therefore, it can be assumed that the influence of testosterone is not direct but indirect, i.e., via a testosterone metabolite or testosterone-induced factors [Testosterone is a crucial steroid hormone. As an important messenger substance, it also interacts with pathogens in the body. It also has a certain influence on plasmodia. For example, in 1991, research showed that host testosterone has a negative effect on the self-healing ability of eatment) , 84. Theeatment) . However factors , 85.A total of about 70 vaccine candidates are currently in various stages of scientific and clinical research. Optimistically, an effective vaccine against malaria is only a matter of time. The novel mRNA vaccines offer a further opportunity to develop effective vaccines. The Tübingen-based company CureVac is already planning the first registration trials . The quePlasmodium and its complex cell structure. Past experience shows that the development of drugs and agents against complex Protozoa is notoriously difficult, time-consuming, and expensive [At this point, it is not really possible to say which way future mRNA vaccines will work and which antigens they will target. This is partly because potential candidates are still in the research phase and companies in particular do not publicly discuss these phases. Another problem is with xpensive , 87. On xpensive , 89.Plasmodium is also such a surface protein. Surface proteins  are major players in evolution [Plasmodium, possess such a compartment: the apicoplast.In the following, some possible antigens will be described. It can be assumed that future mRNA vaccines could target on of these antigens. Among other things, because earlier non-mRNA vaccines already target these proteins. However, despite all this, this part of the article is clearly more speculative than the rest of the text and should be read with this knowledge in mind. What can be assumed with a high degree of certainty is that surface proteins will play a relevant role in development. Surface proteins are crucial for pathogen–host interaction. As exposed structures, they are the first pathogen molecules to come into contact with the host molecules and, therefore, with the host immune response , 91. Thuvolution , 93. KnoPlasmodium berghei, a Plasmodium species that infects rodents. A transgenic parasite strain of P. berghei was used. For the study, sporozoites were injected intravenously at three doses . Parasitemia in the blood was monitored for 14 days. A total of three doses of PfCSP mRNA-LNP1 were used in the study. A 30-µg dose, a 10-µg dose, and a dose containing only LNP1 (which served as a control). Animals were vaccinated twice, 3 weeks apart. Two weeks after the last vaccination, blood samples were collected and analyzed. The high dose achieved a vaccination efficacy of 40%, while the lower dose achieved a vaccination efficacy of 20%. The non-vaccinated control group invariably developed blood-stage infection. The results make it a compelling candidate for further investigation and suggest that CSP-based mRNA vaccination is very likely [The circumsporozoite protein (CSP) is a surface protein consisting of three regions: the N-terminus, a 4-amino acid repeat region, and the C-terminus. While the N-terminus is capable of binding sulfate proteoglycans, the C-terminus possesses a thrombospondin-like type I repeat domain (TSR) . CSP is Plasmodium species [The merozoite surface protein-1 (MSP-1) is another surface protein that occurs in merozoites. MSPs are synthesized at the beginning of the schizogenesis and important in the infection of red blood cells. Located on the merozoite surface, MSPs are anchored to the membrane with glycophosphatidylinositol, a glycolipid. The MSP-1 complex binds to Spectrin, a surface protein of erythrocytes which is important for cell stability. In addition to MSP-1, there are other MSPs. MSP-2 plays a similar role as MSP-1, although its function has not yet been clarified. The roles of MSPs MSP-3, MSP-6, MSP-7 and MSP-9 are also unclear . Vaccine species . This co species . In addi species .In addition to MSPs, Rhs are important for erythrocyte invasion. Rh means reticulocyte-binding homolog. The family includes Rh1, Rh2a, Rh2b, Rh4 and Rh5. Rh5 is the smallest protein of this family. One reason for this is that Rh5 is the only Rh protein that does not have a transmembrane domain. This is unique among Rh proteins . Rh5 reaAlso expressed on the surface is Pfs25. Pfs25 is a sexual stage antigen as it is present on the surface of zygotes and ookinetes. Since antibodies against Pfs25 are capable of completely arresting oocyst development, Pfs25 is of interest for the development of transmission-inhibiting vaccines . MonoclomRNA vaccines have been in development for several years. However, it is only the current COVID-19 pandemic that has practically triggered a medical revolution. Working and highly effective vaccines against SARS-CoV-2 were developed in record time. It is, therefore, not surprising that the responsible companies are expanding their focus to other pathogens. In the future, we will have different mRNA vaccines against different diseases. In addition, one of them will probably be against malaria. This perspective highlights which proteins and antigens are candidate for potential mRNA vaccines. Although the circumsporozoite protein is probably one the best candidates, other antigens also have the potential to form the basis for effective mRNA vaccination. However, even these considerations are highly speculative at the moment. What can be said in conclusion is that a potent vaccination against malaria is currently probably only a question of time. The knowledge gained from the SARS-CoV-2 pandemic and the associated development of novel vaccines (including mRNA vaccines) will have a positive impact on future developments. Even if the first approved vaccines against malaria are not mRNA vaccines.