Asymptomatic malaria infections are important parasite reservoirs and could sustain transmission in the population, but they are often unreported. A community-based survey was conducted to investigate the prevalence and factors associated with asymptomatic malaria infections in a historically high transmission setting in northern Uganda.Using a cross-sectional design, 288 children aged 2–15 years were enrolled and tested for the presence of malaria parasites using rapid diagnostic tests (RDTs) and blood smear microscopy between January to May 2022. Statistical analysis was performed using the exact binomial and Fisher’s exact test with p ≤ 0.05 indicating significance. The logistic regression was used to explore factors associated with asymptomatic malaria infections.Plasmodium falciparum was the dominant species (86%) followed by Plasmodium malariae (5%). Factors associated with asymptomatic malaria infection were sleeping under mosquito net (Adjusted Odds Ratio (aOR) 0.27; 95% CI 0.13–0.56), p = 0.001 and presence of village health teams (VHTs) , p = 0.001. Sensitivity and specificity were higher for the P. falciparum/pLDH RDTs compared to HRP2-only RDTs, 90% (95% CI 86.5–93.5) and 95.2% (95% CI 92.8–97.7), p = 0.001, respectively.Overall, the prevalence of asymptomatic infection was 34.7% (95% CI 29.2–40.5) with the highest observed in children 5–10 years 45.9% (95% CI 35.0–57.0). Gweri village accounted for 39.1% (95% CI 27.6—51.6) of malaria infections. Median parasite density was 1500 parasites/µl of blood. Plasmodium falciparum was the dominant parasite species however the presence of P. malariae and Plasmodium ovale was observed, which may have implication for the choice and deployment of diagnostic tools. Individuals who slept under mosquito net or had presence of functional VHTs were less likely to have asymptomatic malaria infection. P.f/pLDH RDTs performed better than the routinely used HRP2 RDTs. In view of these findings, investigation and reporting of asymptomatic malaria reservoirs through community surveys is recommended for accurate disease burden estimate and better targeting of control.Asymptomatic malaria infections were present in the study population and this varied with place and person in the different age groups. The online version contains supplementary material available at 10.1186/s12936-022-04421-1. Plasmodium falciparum accounts for > 95% of malaria infections in Uganda [The WHO African Region continues to contribute a disproportionately high share of the global malaria burden accounting for 95% of malaria cases . Uganda n Uganda . Asympton Uganda , 9.In Uganda, several studies have reported a high burden of asymptomatic infections in children . MathemaAs part of the efforts to control malaria in this region, the Ministry of Health established a sentinel-surveillance site in Aduku to conduct therapeutic efficacy studies and collect high-quality malaria data to inform control interventions. The HMIS and the mid-term review of the 2015 malaria strategic plan have reported reductions in malaria burden in Aduku, however, these estimates are majorly based on symptomatic individuals, who come for care and treatment at health facilities .In addition to symptomatic cases, the country’s malaria control policy recommends the identification and clearance of parasites in asymptomatic infections as an important intervention for malaria elimination. However, there is no recent data on the burden of asymptomatic parasite reservoirs in communities living in Aduku and burden remains unknown. Asymptomatic infections are important malaria parasite reservoirs which sustain malaria transmission in communities  that comThis was a household community-based cross-sectional study that enrolled participants from a random sample of villages and households in Aduku sub-county. The first stage of sampling was at sub-county level where a list of all parishes in the sub-county was obtained which formed the first sampling frame. Using simple random sampling, four (4) parishes of Apire, Alira, Aboko and Ongoceng were selected. The second stage of sampling was done at the parish where a list of all villages in each of the 4 parishes formed a sampling frame. From each parish, one village was randomly selected. The selected villages were Gweri, Egum, Akwon and Amia A. From each village, a random sample of households was selected from which eligible children were enrolled into the study.The study was conducted in four randomly selected villages and parishes in Aduku sub-county in Kwania district. Aduku is located approximately 282 km north of Kampala city. The geographical positioning/coordinates of Aduku are: 2°01′10.0"N, 32°43′12.0"E . It is traditionally a high malaria transmission area with one of the highest EIR ever reported in the world because of the conducive climatic and environmental conditions optimal for breeding of malaria vectors –16. AdukChildren aged 2–15 years living in the selected parishes and villages in the selected households in Aduku sub-county were enrolled into the study if they met the eligibility criteria. Where children were unable to assent, the care taker provided the required information. Children’s care takers or guardians were administered a study questionnaire to collect additional demographic variables. The rationale for choosing the 2–15 years age group as the study population was based on evidence from recent studies that showed rapid shifts in parasitaemia with higher parasite prevalence seen in older children above 5 years as compared to children under 5 , 20.The study sample size was determined using the Kish and Leslie (1965) formula for cross-sectional surveys  based on the following assumptions. The expected proportion of 13% was assumed for asymptomatic malaria infections in this region based on the Uganda National indicator survey . A standThe inclusion criteria were children aged 2–15 years with an auxiliary temperature of < 37.5 °C who provided assent and whose guardian or caretakers allowed them to participate and provided a blood sample for malaria parasites. Exclusion criteria was presence of signs and symptoms of severe malaria (these were immediately referred to the facility) and those who were presently on treatment for malaria.Data collection was done using a short questionnaire which captured individuals’ details and demographics from consented participants. Permission to enroll eligible children was obtained from the care taker or older children who were able to assent. Each enrolled child provided approximately two drops of blood obtained by finger prick for malaria testing. Malaria testing was done by two different malaria RDTs  and blood smear microscopy for each participant. Results of the RDTs were provided to study participants immediately on site while blood smears were collected, dried and transported to Aduku HC IV malaria sentinel surveillance site for staining and microscopy. Both thick and thin smears were collected on same slide.Blood smears were prepared in the field using blood samples obtained from a finger- prick with both thick and thin smears on same slide. Thick smears were used for parasite detection and quantification while thin smear for species identification. Thin smear was fixed with absolute methanol and thereafter the slides kept in the field in a slide box for no longer than 12 h to avoid auto-fixation. At Aduku sentinel site field laboratory, thick blood smears were stained with 10% Giemsa for 30 min before transportation to the malaria research laboratory in Kampala for examination for the presence of parasitaemia. All slides were cross-checked and re-examined by the WHO certified expert slide readers at the research laboratory. Parasite densities were calculated from thick blood smears by counting the number of asexual parasites and reported as parasites per micro litre of blood. A thick blood smear was considered negative when the examination of 100 high power fields did not reveal asexual parasites. For quality control, all slides were read by two independent microscopists and a third reviewer settled any discrepant readings. The standard WHO procedure for Giemsa staining and reading of blood smears was strictly followed .RDTs were done according to the manufacturer’s instructions. Two malaria RDTs P.f-HRP2/(pLDH) Cat. 05FK90 and P.f-HRP2 Cat. 05FK50  were used in the study. Briefly, a drop of blood was placed in the sample well, followed by dropping the buffer into the buffer well. The buffer lyses the RBCs exposing the target antigen and enhance the flow of the sample along the nitrocellulose strip. Appearance of the test and control indicates presence of parasites while presence of control without test line means absence of parasite antigens. The RDT test procedure were done according to the manufacturer’s recommendations.The study was approved by the Mbarara University Research and Ethics Committee. Additional approval to conduct the study was obtained from the Kwania district health office. All participants provided written consent before enrollment into the study.A total of 291 participants were enrolled into the study. Three (3) samples were excluded leaving 288 for analysis and reporting. The detailed enrolment and study flow chart is illustrated in Fig. The study estimated the proportion of asymptomatic malaria in children 2–15 years in Aduku sub-county in Kwania district. Overall, 52.8% of the enrolled participants were female. The majority of the participants were in the 10–15 years age bracket. Although Amia-A village had the biggest number of participants, the sample was evenly distributed across the study villages. The detailed baseline characteristics are indicated in Table Overall, 34.7% 95% CI 29.2–40.5) of the participants had asymptomatic malaria infection by blood smear microscopy which is significantly higher than the regional parasite prevalence (13%) for Lango region [9.2–40.5 P. falciparum (86.0%). However other malaria species were encountered in this study population, Plasmodium malariae (5%), and Plasmodium ovale (1%) Table A majority of malaria infections were due to Overall, the median parasite load in the study population was 1500 parasites per microlitre of blood. The highest parasite density was observed in children of 5–10 years of age . Most of the high-density infections were seen in Amia A village (median density 1800)  as measure of association. Bivariable and multivariable analysis were done to obtain the crude and adjusted estimates respectively. The factors associated with asymptomatic malaria infection were presence of a village health team aOR = 0.02 (95% CI 0.006–0.45), p = 0.001 and sleeping under a mosquito net the previous night aOR = 0.269 (0.130–0.557), p = 0.001  and 95.2% (92.8–97.7), p = 0.001, respectively. The predictive values followed the same trend and were better for the P.f/pLDH RDTs, 90.9% (87.6–94.2) and 94.7% (92.1–97.3), p = 0.001 for PPV and NPV, respectively  followed by the 10–15 years 30.6% (95% CI 22.5–39.6). Traditionally, children < 5 years carried the highest malaria burden in Uganda due to lower immunity , 31. SimPlasmodium infections have been reported to be missed by standard malaria rapid diagnostic tests when the blood antigen concentration is below the detection threshold [Parasite density is a quantitative method that provides an estimate of malaria parasites in an infected individual. While there are several parasite quantification methods including counting parasites/200 WBC, the plus system and quantitative PCR (qPCR), the WHO recommends the use of blood smear microscopy for quantification and reporting of parasite counts per microlitre of blood assuming a standard WBC count of 8,000 cells for healthy individuals . Using ehreshold .Epidemiologically in terms of person, time and place; high-density malaria infections (> 1000/µl) were disproportionately seen in children 5–10 and 10–15 years and in Gweri village in this study population. The presence of high-density infection in relatively older children (above 5 years) is consistent with what was reported previously in Uganda and Tanzania and may provide additional evidence of shifts of parasitaemia from the < 5 to older children , 38. TheP. falciparum (86%) followed by P. malariae (5%). Plasmodium ovale was present, but in extremely low proportions (1%). Predominance of P. falciparum in this study is similar to what is reported in the MOH national malaria survey (MIS) [-P. falciparum species in these samples is also consistent with the results of 2019 MIS that reported an increase in non-falciparum species, particularly P. malariae and P. ovale, in Uganda [Plasmodium falciparum is the most pathogenic species causing the most aggressive form of malaria  [P. falciparum has implication on the type of RDTs to be deployed in this setting since the current HRP2 tests only detects P. falciparum mono-infection. Non-P. falciparum can cause false negative RDTs in settings where P. falciparum only RDTs are exclusively used [P. falciparum + P. malariae, as well as P. falciparum + P. ovale may have implications on the training and developing competence of laboratory personnel in this setting to be able to report these species in the HMIS.Speciation of plasmodium species is an important malaria epidemiological parameter for understanding a country’s parasite population that eventually informs public health control interventions for malaria. In this study, the dominant species was ey (MIS) . The obsn Uganda , 40. Simn Uganda , 38, 40 n Uganda , 41, 42.malaria) . The imp, malaria infection has been associated with factors related the vector, parasite, human host and the environment. However, in this study only the human host related factors were investigated. A number of studies have reported human behavioural and practices that are known to increase the risk of malaria transmission [p = 0.015) [Epidemiologicallysmission , 31. In smission . The usesmission . Mosquitsmission , 44 and smission . Similarsmission . The red= 0.015) , while i= 0.015) .P. falciparum pLDH RDTs. Although HRP2 RDTs are designed to detect the P. falciparum only, their low specificity (89%) could be attributed mainly due to their inability to detect the non-P. falciparum species observed in these samples [P. falciparum is the predominant parasite species [The study investigated the field performance of several diagnostic tools used for malaria testing during the survey in this setting. Accurate diagnosis of malaria parasites is important not only for administering correct treatment but also for surveillance and accurate estimation of disease burden to inform malaria control programme strategies. However, the diagnostic performance of RDTs can deteriorate and requires periodic monitoring –49. In t samples  and simi samples . HRP2-on species . However species , 52.P. falciparum species reporting infected individuals as negative . This observation is consistent with the results of previous studies that showed the occurrence of false-negative HRP2 RDTs in non-P. falciparum clinical samples [P. falciparum species in the study setting suggests that RDTs that target alternative antigens (other than HRP2 only) may be more appropriate for future use in case management and surveillance in this and other similar settings [The decreased specificity of the HRP2 RDTs observed in this study suggests that HRP2-only RDTs can potentially miss detection of non- samples . The presettings . Previousettings –57. Howesettings , 58–61. settings , 62, 63.settings , 64, 65.settings .Other known causes of false-negative RDTs include, transport and storage conditions, and user-related factors , 62. HowThe study had limited geographical coverage and, therefore, generalizability may be not possible. Although blood smear microscopy was used as gold standard as recommended by the WHO , 43, thePlasmodium falciparum was the dominant parasite species however the presence of P. malariae and P. ovale was observed which may have implication for the choice and deployment of diagnostic tools. Individuals sleeping under mosquito net and had presence of functional VHTs were less likely to have asymptomatic malaria infection. Plasmodium falciparum pLDH RDTs performed better than that the routinely used test HRP2 RDTs. In view of these findings, investigation and reporting of asymptomatic malaria reservoirs through community surveys is recommended for accurate disease burden estimate and better targeting of control interventions.Asymptomatic malaria infections were present in the study population and this varied with place and person in the different age groups. Additional file 1. The detailed datasets analyzed are uploaded as additional data file 1.


Studies have demonstrated the ototoxic effects of antimalarial drugs in individuals who receive these drugs, but little is known regarding the toxicity of these drugs in the newborn auditory system when administered to the mother receive the drug during pregnancy.To verify the incidence of hearing loss in neonates who have no other associated risk indicators, born to mothers treated for malaria during pregnancy.A retrospective, quantitative cohort study was developed at Hospital de Base Dr. Ary Pinheiro and Clínica Limiar, both located in the municipality of Porto Velho (Rondônia). The sample consisted of 527 newborns divided into two groups: exposed to antimalarials drugs during pregnancy group (n = 32) and non-exposed group (n = 495). Data collection took place from September 2014 to December 2015, through an interview with the mothers and/or guardians of the newborn, through the newborns’ and the mothers’ records, and the neonatal hearing screening database of the above-mentioned institutions.Plasmodium vivax was the most frequent, and was similarly distributed among the gestational trimesters, and chloroquine was the most commonly used antimalarial drug treatment more often given during the third trimester; these findings did not show any influence on the audiological findings of the studied neonates.All the neonates in the exposed group, assessed through the recording of transient otoacoustic emissions associated with the automated brainstem auditory evoked potential test, underwent neonatal hearing screening in the first examination. Among the newborns in the non-exposed group, 30 showed failure and were retested. Of these, one continued to fail and was referred for diagnosis, in whom the results showed to be within the normal range. Among the neonates of the exposed group, infection with The present study did not identify any cases of hearing loss in neonates born to mothers who used antimalarial drugs during gestation. Malaria is the most important endemic infectious disease in the Brazilian Amazon region, due to its wide dissemination in the area, high incidence, impact on morbidity and mortality and difficultly to control.Of the nine states that constitute the Legal Amazon region , Rondônia ranks second and third in recorded cases of malaria between 2002 and 2011.In 2006, the municipalities of Cruzeiro do Sul (AC), Manaus (AM) and Porto Velho (RO) accounted for 22.59% of the total cases of malaria in the Amazon region.Plasmodium falciparum; early and correct diagnosis and timely treatment are the most appropriate means to reduce malaria severity and lethality.The clinical picture of malaria can be classified as mild, moderate or severe according to the parasite species, the number of circulating parasites, the duration of disease and the level of immunity acquired by the patient.Among the complications of maternal malaria during conception and delivery, studies have recorded cases of miscarriage,11Plasmodium vivax and 3 cases of mixed malaria .The number of positive slides in pregnant women in the municipality of Porto Velho in 2010 totaled 331 cases combining rural and urban areas, including 48 cases of P. falciparum, 280 cases of Studies have demonstrated the ototoxic effects of antimalarials in individuals who use these drugs;Studies have reported that when chloroquine crosses the placenta, it may be ototoxic to fetuses.A study carried out in 2004, confirmed 12 cases of hearing loss in children whose mothers had been treated with antimalarials during pregnancy. All the children were diagnosed with sensorineural hearing loss, seven of them profound and five between moderate and severe.In Porto Velho (RO), there were two cases (11.8%) of hearing loss in children from zero to three months of age whose mothers used antimalarials during pregnancy. Both children were diagnosed with unilateral mild sensorineural hearing loss.A study carried out in the NHS program of the referral public hospital of the state of Rondônia showed that the neonates of mothers treated for malaria during pregnancy had a percentage of failure in the NHS program of 6.7% that was 5.64 times higher than the newborns (NBs) of untreated mothersThe need to evaluate the effect of antimalarial use in the gestational period on the hearing of the newborns without the possibility of the influence of other RIHL justifies the present study. Based on the above, the aim was to verify the frequency of hearing loss in neonates of mothers treated for malaria during pregnancy, without other associated RIHL, as well as to characterize the exposed group (EG) and the non-exposed group (NEG) to antimalarials in the gestational period regarding the variables: age, origin and maternal schooling, as well as gestational age (GA) and birth weight; to characterize the EG regarding the type and trimester of the malaria infection and the type of antimalarial drug used and the gestational trimester when the medication was administered.This is an epidemiological, observational, analytical, historical cohort study, evaluated and approved by the Research Ethics Committee (REC) with human subjects under number 314,929. The present research was carried out at Hospital de Base Dr. Ary Pinheiro (HBAP) and at Clínica de Avaliação e Reabilitação da Audição — Limiar, both located in the city of Porto Velho (RO).All newborns in this study whose mothers did not receive antimalarial treatment during the gestational period were screened with Transient Evoked Otoacoustic Emissions (TEOAE), and neonates who failed the test were retested within 15 days. The newborns whose mothers received antimalarial drugs, as well as those who did not receive them, but failed the evaluation with TEOAE, were screened using the combined technique . Data collection for this study occurred between September 2014 and December 2015.The following eligibility criteria were established for case selection: newborns of Brazilian mothers, born at HBAP in rooming-in care, without RIHL, or only with the antimalarial factor during pregnancy, submitted to all phases of the NHS program at the time of data collection and have performed all recommended steps for neonatal hearing screening program,The sample was estimated based on the mean number of newborns in rooming-in care screened at the HBAP during the data collection period, which is 3600. Adopting a 5% error and a level of significance of 95%, a minimum sample size of 348 newborns was estimated.Prior to hospital discharge, 734 mothers or guardians of newborns in rooming-in care admitted to the HBAP at the time of data collection were approached, who agreed to participate in the study by authorizing access to the medical records of the mother and the newborn. Of these, 173 were excluded because they had RIHL, 25 because they did not perform all the recommended steps for the NHS programs  and 9 because the mothers did not undergo prenatal care.Based on the above, the sample comprised 527 newborns, who were divided into two groups, namely: exposed group (EG) and non-exposed group (NEG). The first group consisted of 32 newborns of mothers who used antimalarial drugs during the gestational period and the second of 495 newborns with no risk of hearing loss in childhood.Aiming at isolating the variable “use of antimalarials in the gestational period” and to avoid the presence of confounding variables, only newborns without RIHL were included in NEG, whereas the EG included newborns born to mothers who used antimalarials during the gestational period with no other associated RIHL.Of the 32 neonates that comprise the EG, 17 were females (53.1%) and 15 males (46.9%) and of the 495 neonates in the NEG, 251 were females (50.7%) and 244 (49.3%) were males.The newborns from the EG had a mean GA of 38.4 weeks and a mean birth weight of 3131 g, while the newborns from the NEG had a mean GA of 38.6 weeks and a mean birth weight of 3229 g.Data collection started with an interview with the newborns’ parents or guardians to obtain socioeconomic and demographic data, family history of hearing loss in childhood, and confirm the information that was subsequently obtained from the medical records, such as the occurrence of congenital infections and prenatal care and, in the cases of mothers who had gestational malaria, information on the type of infection, period of occurrence, drug treatment, and the period during which the medication was administered  was obtained.In addition to the interview, the newborn’s and the mother’s medical files, the HBAP NHS database, the Clínica Limiar database, the GN Otometrics MADSEN AccuScreen® portable device, the newborn’s vaccination card, the pregnant woman's card and data collection form were used for data collection.p ≤ 0.05).All compiled data were recorded in the data collection form, in order to guarantee the organization and quality of the data collection, tabulated in Excel spreadsheets and submitted to statistical tests with a significance level of 5% , as well as to compare these variables between the groups. To analyze the distribution of the qualitative variables  as well as to compare such variables between the groups, the statistical test of equality of two proportions was used. This statistical test was also used to characterize the subjects of the EG, regarding the type and gestational trimester of malaria infection, as well as to verify the different antimalarial drugs used during the gestational period, as well as the gestational period of their administration, in addition to comparing the TEOAE results, according to the ear, between the groups and compare the results of the NHS tests between the groups.In addition to these analyses, it was possible to calculate the relative risk of the newborns from both groups to fail the TEOAE in the left ear, whereas it was not possible to perform the same analysis regarding the TEOAE in the right ear and the final result of the NHS, since no failures were obtained in the right ear at the TEOAE and in the final result of the NHS in the EG.We documented infection in five mothers who were infected in more than one gestational period; the gestational period of malaria infection in the mothers treated with Coartem (artemether + lumefantrine) and Quinine + Clindamycin; the number of mothers treated for malaria after the infectious period and the treatment received according to the gestational trimester (one or more trimesters).Finally, the results of the audiological evaluation of the newborns referred for diagnosis were described.p = 0.140).The age of the mothers who had malaria during the gestational period ranged from 14 to 39 years, with a mean of 23.3 years and mothers who were not affected by the disease ranged from 14 to 43 years, with a mean of 25.1 years. These values, when compared, did not show any statistically significant difference (p < 0.001). Among the mothers of the newborns from the EG, 24 come from the capital of Rondônia, six from the countryside of the state and two from the countryside of the neighboring state (Amazonas). Among the mothers of the newborns from the NEG, 336 were from Porto Velho, 154 from the countryside of the state and 5 from the countryside of the neighboring state (Amazonas).It was demonstrated, in both groups, that the significant majority of the mothers came from Porto Velho (RO) (p < 0.0001); Complete High School — CHS (p = 0.044).Regarding the level of schooling, most mothers from the EG  reported not having finished Elementary School, a significant finding when compared to the other levels of schooling: Complete Elementary School — CES, Incomplete High School — IHS, Incomplete College/University — IC/U and Complete College/University — CC/U (p = 0.250), indicating a significant difference when compared to other levels of education .Among the NEG mothers, the majority reported having completed high school , a finding that did not differ statistically only from the number of mothers who did not complete elementary school , with the treatment performed with quinine associated with clindamycin, and primaquine .Table 3DP. falciparum, with two of them infected in the second trimester and one in the third trimester of pregnancy. Of the two women treated with quinine associated with clindamycin, both were infected in the first trimester, but one was infected by P. vivax and the other by P. falciparum.The three mothers treated with Coartem (artemeter + lumefantrine) were infected by P. falciparum reported having been treated with chloroquine, one of whom used this antimalarial drug in combination with primaquine.Moreover, three mothers infected with The drug treatment with antimalarials was more frequent in the third trimester, and this result was significant when compared to antimalarial administration in the first trimester of pregnancy . FourteeThree mothers (9.38%) underwent treatment only in the first trimester, whereas another 3 mothers (9.38%) underwent treatment only in the second trimester, and 8 mothers (25%) underwent treatment only in the third trimester. One mother (3.12%) reported having used antimalarial drugs in the first and second gestational trimesters, 2 (6.25%) reported treatment in the first and third trimesters, 7 (21.87%) reported treatment in the second and third trimesters and 8 (25%) were treated for malaria during the three gestational trimesters.When the isolated TEOAE results were analyzed according to the tested ear, it was verified, in the first test, that no EG newborn failed during the right ear evaluation, whereas one newborn failed the left ear test. On the other hand, 21 of the newborns of the NEG failed the TEOAE in the right ear and 29 in the left ear. These findings showed statistical significance when the intragroup comparison was performed, but no statistically significant difference was observed when comparing the findings between the groups . No assoThere was a higher incidence of TEOAE failure in the left ears of newborns from the NEG, indicating that the use of antimalarials in the gestational period may protect the hearing of newborns . HoweverThe newborns from the EG who showed TEOAE failure in the left ear were not retested, since they showed a satisfactory response in the A-BAEP test, thus “passing” the NHS.Thus, only the newborns who showed “failure” in the first test and belonged to the NEG, which were evaluated only through TEOAE, underwent the retest. Of the 30 newborns who underwent TEOAE retest, only one patient continued to show “failure”  and was then referred for diagnostic evaluation.All newborns from the EG, evaluated through the combined technique (TEOAE + A-BAEP), passed the NHS immediately after the first examination. Of the 495 newborns that comprised the NEG, evaluated only through TEOAE, as they were considered of low risk for hearing loss, 33 showed “failure” at the first test, and of these, three underwent A-BAEP and passed, with retesting not being necessary.Of the 30 retested newborns, 53.3% (n = 16) were retested before hospital discharge and 46.6% (n = 14) were reassessed 15 days after the first test, i.e., after hospital discharge. After retesting, only one newborn persisted showing “failure” at the test, which was bilateral.Thus, of the 32 newborns from the EG, all passed the NHS (100%) and of the 495 from the NEG, 494 passed the NHS (99.8%), with the newborn that failed being referred to diagnostic evaluation.This neonate came for diagnostic evaluation at 22 days of age and showed absence of diagnostic TEOAE in both ears; presence of waves I, III and V in the diagnostic BAEP, with absolute and interpeak latencies within the normal range, demonstrating integrity of the auditory pathway to the high brainstem; electrophysiological threshold at the frequency of 2000 Hz within normality (30 dB) and normal mobility of the ossicular-tympanic system (type A tympanometric curve), bilaterally. Considering these results, and the fact that this newborn did not have RIHL, he was discharged from the NHS program and the mother was instructed regarding the auditory and linguistic development of the child.Therefore, none of the 527 newborns who participated in the present study  were diagnosed with hearing loss.The present study did not demonstrate any cases of hearing loss in neonates born to mothers who used antimalarial drugs  during pregnancy. This finding corroborates that found by Silva (2014), which characterized the NHS program of the hospital where this study was developed, which verified that although the occurrence of malaria during the gestational period is the infection with the most frequent risk of vertical transmission in the studied population, none of the infants diagnosed with hearing impairment had a history of gestational malaria.Malaria during pregnancy was also a risk indicator with a significant occurrence in neonates under rooming-in care, in a study carried out with newborns from private health units in the city of Porto Velho (RO); however, the neonates exposed to gestational malaria did not have hearing loss.Another study, carried out in the same municipality, diagnosed 15 newborns with hearing impairment (2/1000 newborns), but no cases of antimalarial use were detected in the gestational period among the diagnosed neonates.On the other hand, other studies verified hearing loss in children exposed to antimalarials in the gestational periodTherefore, there is still no evidence that “antimalarial use during pregnancy” constitutes a risk factor for hearing loss in neonates.Moreover, the present study did not show a significant difference in GA and in the weight of neonates from both groups , with these findings being quite similar to those found by other researchers.It is believed that the mothers of the present study who had malaria during pregnancy received an early diagnosis and timely treatment, with no alterations in the course of gestation, as well as in GA and birth weight.P. falciparum infection, treated with quinine and 130 episodes of P. vivax infection, treated with chloroquine, which found that there was no increase in rates of congenital abnormalities, fetal death or low birth weight. According to the researchers, these results suggest that the therapeutic doses of chloroquine and quinine are safe.11This result also confirms the findings of a study carried out in Thailand with 300 pregnant women, totaling 376 episodes of malaria, of which 246 were of Another hypothesis that may justify the absence of alterations during the course of gestation and, consequently, in the GA and in the weight of the newborns of the present study is the fact that the identified malarial episodes were not classified as severe or complicated ones.p = 0.007); showing significantly lower birth weight in the newborns from the group exposed to malaria during gestation.In contrast, a study carried out in Ibadan, Southwest Nigeria, found an association between maternal malaria during pregnancy and the newborn’s birth weight , which is recommended for pregnant women with P. falciparum malaria in the second and third trimesters.P. falciparum malaria in the second and third gestational trimesters.Of the seven cases of P. vivax and the other with P. falciparum, both in the first trimester of pregnancy, the treatment recommended by the Ministry of Health for cases of severe and complicated malaria caused both by P. falciparum and P. vivax in the first gestational trimester.Only two mothers were treated with quinine together with clindamycin, one infected by P. falciparum infection with chloroquine, which is contraindicated because this parasite is resistant to the aforementioned drug.P. falciparum in the first trimester of pregnancy, although primaquine is contraindicated because it can result in fetal hemolysis.P. falciparum, but it is contraindicated during pregnancy due to undesirable effects on the fetus, such as methemoglobinemia.It was also reported that three mothers were treated for P. falciparum were treated with chloroquine, a drug that is not effective against this Plasmodium due to its resistance to the medication.Although most of the treatments performed are in agreement with recommendations of the Ministry of Health,In the present study, drug treatment with antimalarial drugs was more frequent in the third trimester, a result that corroborates findings from other studiesAs for the auditory evaluation of the neonates, the first test showed a higher number of failures in the left ear, as previously shown in other studies with newborns,45All newborns exposed to antimalarial drugs in the gestational period were screened using TEOAE associated with A-BAEP, as recommended for neonates at high risk for hearing loss.In contrast to the results obtained in the present study, Silva et al. demonstrated a 6.7% failure rate in the NHS of newborns whose mothers had been treated for malaria during pregnancy, showing they were 5.64 times more likely to fail in NHS than the NB of mothers not treated for gestational malaria.We suggest the need for new studies, with a larger number of subjects, a situation that was not feasible in this study because of the changes that occurred in the HBAP NHS program at the time of data collection, which currently screens all newborns only through OAE.Moreover, performing a case-control study might also be appropriate because hearing loss, as well as malaria infection, are not frequent. However, in this situation, case-control studies could have a bias due to the fact that the neonates in the group (with hearing loss) might show other RIHL associated with treatment for malaria during pregnancy and hinder establishing the association between hearing loss and the use of antimalarials during pregnancy, as the alterations observed in this group may be due to the other RIHL observed in the neonates, thought to have occurred in other previous studies.25It is believed that the findings of the present study, related to GA, newborn birth weight, as well as the absence of auditory alterations in neonates exposed to antimalarial drugs in the gestational period, are explained by the adequate and timely treatment, as proposed by the Ministry of Health.We also suggest that the hearing of these newborns be monitored and studies with children and adolescents exposed to antimalarial drugs in the gestational period, be developed aiming to investigate the occurrence of progressive hearing loss in this population. A study of 725 children and adolescents from Mozambique who received antimalarials, identified 12 subjects, with a mean age of 3.2 years who received antimalarials in the gestational period, and were identified with sensorineural hearing loss, seven of them with profound and five with moderate to severe hearing loss.The present study of neonates with no other associated RIHL did not identify any cases of hearing loss in neonates born to mothers who used antimalarial drugs  during pregnancy, with all neonates from the EG successfully going through NHS tests with TEOAE and A-BAEP since the first test.Based on these facts, it is necessary to develop studies that aim to monitor the hearing of children exposed to antimalarial drugs in the gestational period, preferably without other RIHL, aiming to investigate the occurrence of progressive hearing loss in this population.The authors declare no conflicts of interest.


P. falciparum chimeric protein that contains a fragment of the sexual-stage Pfs48/45-6C protein genetically fused to GMZ2, an asexual-stage vaccine construction consisting of the N-terminal region of the glutamate-rich protein (GLURP) and the C-terminal region of the merozoite surface protein-3 (MSP-3). Previous studies showed that GMZ2.6c is widely recognized by antibodies from Brazilian exposed individuals and that its components are immunogenic in natural infection by P. falciparum. In addition, anti-GMZ2.6c antibodies increase with exposure to infection and may contribute to parasite immunity. Therefore, identifying epitopes of proteins recognized by antibodies may be an important tool for understanding protective immunity. Herein, we identify and validate the B-cell epitopes of GMZ2.6c as immunogenic and immunodominant in individuals exposed to malaria living in endemic areas of the Brazilian Amazon. Specific IgG antibodies and subclasses against MSP-3, GLURP, and Pfs48/45 epitopes were detected by ELISA using synthetic peptides corresponding to B-cell epitopes previously described for MSP-3 and GLURP or identified by BepiPred for Pfs48/45. The results showed that the immunodominant epitopes were P11 from GLURP and MSP-3c and DG210 from MSP-3. The IgG1 and IgG3 subclasses were preferentially induced against these epitopes, supporting previous studies that these proteins are targets for cytophilic antibodies, important for the acquisition of protective immunity. Most individuals presented detectable IgG antibodies against Pfs48/45a and/or Pfs48/45b, validating the prediction of linear B-cell epitopes. The higher frequency and antibody levels against different epitopes from GLURP, MSP-3, and Pfs48/45 provide additional information that may suggest the relevance of GMZ2.6c as a multi-stage malaria vaccine candidate.The GMZ2.6c malaria vaccine candidate is a multi-stage  Plasmodium parasites, remains a major public health problem worldwide. In 2021, 247 million cases and 619.000 deaths due to malaria were estimated globally, most of them caused by P. falciparum [P. falciparum malaria in all endemic countries [Anopheles to available insecticides [P. falciparum transmission [Malaria, an infectious disease caused by ountries ,4,5,6, asmission . HoweverP. falciparum chimeric protein that contains a fragment of the sexual-stage Pfs48/45-6C protein genetically fused to GMZ2, an asexual-stage vaccine construct consisting of conserved domains of glutamate-rich protein (GLURP) and merozoite surface protein-3 (MSP-3) [Pfs48/45 is expressed on the surface of gametocytes and gametes, playing a central role in fertilization [P. falciparum growth in vitro by opsonic phagocytosis [Pfs48/45 can recognize extra-erythrocytic gametes, reducing transmission by preventing fertilization and sporogonic development in the mosquito midgut [The GMZ2.6c malaria vaccine candidate is a multi-stage  (MSP-3) . GLURP i (MSP-3) ,11, MSP- (MSP-3) ,13, and lization . Both GLocytosis  and antiocytosis ,17,18 coo midgut .Pfs48/45) are immunogenic in natural infection by P. falciparum. Moreover, anti-GMZ2.6c antibodies seem to increase with exposure to malaria infection and may contribute to parasite immunity [P. falciparum chimeric protein GMZ2.6c in individuals exposed to malaria living in the Brazilian Amazon.Previous studies showed that GMZ2.6c protein was widely recognized by naturally acquired antibodies from individuals of Brazilian endemic areas and that its components , a medium-risk area situated in Amazonas State. Cruzeiro do Sul, Mâncio Lima, and Guajará registered 5.447, 1.432, and 674 cases in 2016, accounting for 52.6% of all P. falciparum infections in Brazil. Due to the increase in P. falciparum cases in other municipalities, this number decreased to 24.1% in 2018, with 2.915, 1.719, and 452 P. falciparum infections in Cruzeiro do Sul, Mâncio Lima, and Guajará, respectively [A cross-sectional cohort study was carried out from June to August 2016 and 2018 in three malaria-endemic areas of the Brazilian Amazon: Cruzeiro do Sul (07°37′50″ S/72°40′13″ W) and Mâncio Lima (07°36′49″ S/72°53′47″ W), both high-risk areas situated in Juruá Valley, Acre State, the main ectively .27−500 (n = 258), MSP-3155–249 (n = 170), and/or Pfs48/45291–428 (n = 108) recombinants  who had neither history of malaria nor contact with malaria transmission areas were included in our study (Rio de Janeiro controls).Serum samples from 303 malaria-exposed individuals identified as responders to GLURPmbinants , as prevDonors providing informed consent answered an epidemiological survey. To evaluate the degree of malaria exposure, subjects responded to questions related to personal data such as age, time of residence in the endemic area, number of previous malaria episodes, time elapsed from the last infection, use of malaria prophylaxis, and presence of symptoms.Venous peripheral blood (20 mL) was collected into Heparin or EDTA tubes for antibodies analysis or molecular diagnosis, respectively. The plasma was stored at −20 °C, and the pellets, containing peripheral blood cells collected into EDTA tubes, were mixed with equal volumes of a cryopreservation solution (0.9% NaCl/4.2% sorbitol/20% glycerol) and were stored at −70 °C until use.Plasmodium sp.) and species  was performed in all samples, as previously described [P. vivax and/or P. falciparum at the time of blood collection were subsequently treated by the chemotherapeutic regimen recommended by the Brazilian Ministry of Health [Thin and thick blood smears were examined for identification of malaria parasites by a technician experienced in malaria diagnosis from the Laboratory of Malaria Research (Fiocruz), which is the headquarters of the CEMART , a reference center for malaria diagnosis in the Extra-Amazonian Region for the Brazilian Ministry of Health. Thick blood smears from all the subjects were stained with Giemsa, and a total of 200 microscopic fields were examined under a 1000-fold magnification. Thin blood smears of the positive samples were examined for species identification. The parasite density was determined by counting the parasites in a predetermined number of white blood cells in the thick blood films, and the number of blood parasites per milliliter was calculated . To increscribed . Positivf Health .Pfs48/45291–428 (UniProt: Q8I6T1) was carried out using the program BepiPred 1.0 [The prediction of linear B-cell epitopes of Pred 1.0 . This me155–249  and 13 synthetic peptides of GLURP27–500 , previously described [Pfs48/45291–428 (Pfs48/45a and Pfs48/45b) identified by prediction of linear B-cell epitopes were used  in citrate-phosphate buffer , pH 5.0, were added to each well, the plates were incubated for 5 min at room temperature in the dark, and then 50 µL/well of 2N H2SO4 (Sigma) were added. Optical density was identified at 492 nm using a SpectraMax 250 ELISA reader . Samples of non-endemic controls obtained from 5 individuals of the laboratory staff (Rio de Janeiro controls) were used to establish the normal range for each assay. The cut-off values were determined as the mean optical density (OD) plus 3 standard deviations of the Rio de Janeiro controls. The results were expressed as the reactivity index (RI), which was calculated by the OD mean of each tested sample divided by the cut-off value. Subjects were scored as responders if the RI against each epitope was higher than 1.0.Microtiter 96-well plates  were coated with 5 µg/mL of each synthetic peptide in carbonate-bicarbonate buffer, pH 9.6, at 100 µL/well overnight at 4 °C. The plates were washed with phosphate-buffered saline 0.05% Tween20 (PBST), and the uncoated sites were blocked with 5% powdered milk containing PBST for 1 h at 37 °C. Serum samples diluted 1:100 in dilution buffer (1% powdered milk containing PBST) were added in duplicate wells, and plates were incubated for 1 h at 37 °C. The plates were washed, 100 µL of peroxidase-conjugated mouse anti-human IgG  1:1000 in dilution buffer was added, and the plates were incubated for 1 h at 37 °C. To detect specific IgG subclass, plates were incubated with peroxidase-conjugated mouse anti-human IgG1, IgG2, IgG3, and IgG4  1:1000 in dilution buffer for 1 h at 37 °C. After washing, 100 µL of a solution of 0.4 mg/mL orthophenylenediamine  and Hp-value of ≤0.05 was considered statistically significant.Data were stored in the Epi-Info version 6 data bank system  and analyzed using Epi-Info version 6 and GraphPad Prism  software programs. Normality tests were performed in all variables using the one-sample Kolmogorov–Smirnov test. The chi-square analysis was applied to compare the prevalence among groups. Kruskal–Wallis, followed by uncorrected Dunn’s test (for multiples comparisons) or Mann–Whitney test (comparisons between two groups), was used to analyze differences in distributions of continuous numerical variables. The Spearman rank coefficient test was used to analyze the correlation between variables. A two-sided Plasmodium species, previous episodes were attributed only to P. falciparum (12 cases), to P. vivax (50 cases), or both parasites (199 cases). The number of reported past malaria episodes varied greatly among participants, ranging from 0 to 100 . The time elapsed since the last malaria infection ranged from 0 to 684 months . At the time of blood sampling, 145 individuals (47.8%) presented symptoms that started 1 to 30 days earlier , with headache, fever, and chills the most frequent ones. A total of 135 individuals (44.5%) had detectable parasites, 53 (17.5%) were infected with P. falciparum , and 82 (27%) were infected with P. vivax .The studied population was composed of 303 individuals living in three malaria-endemic areas of the Brazilian Amazon . The pop27–500  and MSP-3155–249  previously identified as B-cell epitopes to validate their immunodominance in the studied population were selected. The frequencies of individuals with IgG antibodies that recognized at least one of the peptides were 70.9% and 64.7% for GLURP and MSP-3, respectively. The frequency of IgG antibody response varied depending on the peptide, showing that P11 from GLURP27–500 and MSP-3c and DG210 from MSP-3155–249 were preferentially recognized by antibodies from exposed individuals  and MSP-3155–249  were selected. The results showed that epitopes P1, P3, P11, and S3 of GLURP and MSP-3b, MSP-3c, and DG210 of MSP-3 were preferentially recognized by IgG1 and IgG3 cytophilic antibodies, while epitopes P4 of GLURP and MSP-3a of MSP-3 were preferentially recognized by antibodies of the IgG1 subclass. No difference was observed between IgG1, IgG2, IgG3, and IgG4 antibody levels in responders to P3, P4, MSP-3a, MSP-3b, and MSP-3c. However, higher IgG1 than IgG3 antibody levels against P1 and IgG1 than IgG2, IgG3, and IgG4 antibody levels against P11 were detected. In addition, IgG1 and IgG3 were higher than IgG4 antibody levels against S3 and IgG2 antibody levels against DG210 . In addition, IgG3 antibody levels against DG210 were associated with the number of previous malaria episodes , while IgG1 antibody levels against P3 were negatively correlated with time elapsed since the last malaria episode .IgG1 antibody levels against P1 and P11 were positively correlated with age and time of residence in malaria-endemic area (P1: Pfs48/45291–428 (Pfs48/45a and Pfs48/45b) and selected for experimental validation. Most responders to Pfs48/45291–428 recombinant protein presented detectable IgG antibodies against epitopes Pfs48/45a and/or Pfs48/45b. A higher frequency of individuals presenting IgG antibodies against Pfs48/45b than Pfs48/45a was observed. However, low IgG antibody levels were detected in epitopes, without differences between Pfs48/45a and Pfs48/45b  and IgG1 than IgG2, IgG3, and IgG4 antibodies against Pfs48/45b . No difference was observed in IgG1, IgG2, IgG3, and IgG4 antibody levels against Pfs48/45 epitopes.The analyses of IgG subclasses were performed in all samples with detectable IgG antibodies against 27–500, MSP-3155–249, and Pfs48/45291–428 epitopes were compared between non-infected and infected by P. vivax and P. falciparum individuals. The results showed higher frequencies of individuals responders to P7 and P8 infected by P. falciparum when compared with P. vivax-infected individuals. Responders to S3 epitope infected by P. vivax were less frequent than non-infected and P.falciparum-infected individuals. Individuals infected by P. falciparum presented a higher frequency of response to MSP-3a, MSP-3b, and MSP-3c epitopes than non-infected and P.vivax-infected individuals, while the frequency of responders to DG210 infected by P. falciparum was higher than non-infected individuals  that appears to be targeted by cross-reactive antibodies . SimilarThe linear peptide containing the amino acid sequence of DG210 from MSP-3 was also shown to be widely recognized by high levels of naturally acquired IgG antibodies. Interestingly, similar frequency and antibody levels to peptide MSP-3c were found, suggesting that antibody response against DG210 may primarily target this epitope in the studied population. Many seroepidemiological studies demonstrated distinct profiles of antibody response to peptides derived from MSP-3CT. Peptide MSP-3b is the major B-cell epitope in hyperimmune individuals ,35, whilP. falciparum blood-stage antigens, promoting Fc-mediated effector functions as antibody-dependent cellular inhibition (ADCI) [It is widely accepted that cytophilic IgG1 and IgG3 isotypes are the main mediators of the protective humoral response against n (ADCI) ,51, opson (ADCI) ,53, antin (ADCI)  and compn (ADCI) ,56. In cn (ADCI) . In our n (ADCI) ,58 and in (ADCI)  associatn (ADCI) . Howevern (ADCI) ,61.P. falciparum infections and possibly maturation of the immune system over time [Correlation analysis of the immune response and epidemiological data showed that IgG1 antibody levels against GLURP epitopes P1 and P11 were positively correlated with age and time of residence in malaria-endemic area, while IgG3 antibody levels against MSP-3 epitope DG210 were associated with the number of previous malaria episodes. In addition, IgG1 antibody levels against GLURP epitope P3 were negatively correlated with time elapsed since the last malaria episode. These may reflect the cumulative exposure of these individuals to ver time  but did Pfs48/45 antigen is one of the most well-characterized antibody targets of P. falciparum gametocytes. Several studies showed that antibodies against Pfs48/45 displayed a transmission reduction activity in the standard membrane-feeding assay (SMFA) [Pfs48/45 have strong transmission block potential in genetically diverse parasite strains and multiclonal infections [Pfs48/45 have been described. Here, we identified two potential linear B-cell epitopes (Pfs48/45a: Pfs48/45358–366 and Pfs48/45b: Pfs48/45382–391) of Pfs48/45-6c domain using in silico BepiPred algorithm [Pfs48/45291–428 recombinant protein (68.5%) presented detectable IgG antibodies against Pfs48/45a and/or Pfs48/45b peptides, validating the prediction of linear B-cell epitopes. These frequencies are comparable to those found for GLURP27–500 (70.9%) and MSP-3155–249 (64.7%) peptides in this study, as well as other P. falciparum antigens [Pfs48/45b was observed, despite no difference between the antibody levels against both epitopes. Interestingly, antibody levels against Pfs48/45a and Pfs48/45b were similar to those against Pfs48/45291–428 recombinant protein [Pfs48/45-6c is conformational, and the production of a non-properly folded protein did not elicit functional antibodies in mice [Pfs48/45 has revealed that a larger number of antibodies with no transmission-blocking potential recognize the 6c domain of Pfs48/45 [The y (SMFA) ,63, and fections ,65. Tranfections , and no lgorithm . Most ofantigens ,68,69. A protein . These fRP27–500 .9% and M in mice . FurtherPfs48/45 . AdditioPfs48/45 or its binding partners [Studies have revealed that the high transmission-blocking efficacy of the monoclonal antibody 85RF45.1 is due to its ability to block the normal function of partners , and sevpartners .Specific antibodies against sexual-stage antigens are also related to age and serve as markers of exposure, increasing according to gametocyte densities ,74,75, b291 were widely recognized by antibodies from individuals living in endemic areas of Brazil. Combined immunoinformatic and experimental approach strategies identified several linear B-cell epitopes from GLURP27–500, MSP-3155–249, and Pfs48/45291–428 fragments that composed GMZ2.6c, providing additional information that may suggest its relevance as a multi-stage malaria vaccine candidate. Further studies are needed to assess the potential of specific antibodies against GMZ2.6c epitopes in the inhibition of P. falciparum growth and transmission-blocking activity.In conclusion, the data presented here showed that P11 from GLURP, MSP-3c, and DG210 from MSP-3, and the predicted epitopes Pfs48/45a and Pfs48/45b from Pfs48/45


P. falciparum infection and development of severe malaria. In recent years, malaria control has been threatened by COVID-19 pandemic and by the emergence, in Northern Uganda, of both resistance to artemisinin derivatives and to sulfadoxine-pyrimethamine.Uganda accounts for 5% of all malaria cases and deaths reported globally and, in endemic countries, pregnancy is a risk factor for both acquisition of In this facility-based, prospective, observational study, pregnant women will be recruited at antenatal-care visits and followed-up until delivery. Collected data will explore the incidence of asymptomatic parasitemia and malaria-related outcomes, as well as the attitudes towards malaria prevention, administration of intermittent preventive treatment, healthcare seeking behavior and use of insecticide-treated nets. A subpopulation of women diagnosed with malaria will be recruited and their blood samples will be analyzed for detection of genetic markers of resistance to artemisinin derivatives and sulfadoxine-pyrimethamine. Also, to investigate the impact of COVID-19 on malaria care among pregnant women, a retrospective, interrupted-time series will be conducted on at the study sites for the period January 2018 to December 2021.The present study will explore the impact of COVID-19 pandemic on incidence of malaria and malaria-related adverse outcomes, along with the prevalence of resistance to artemisinin derivatives and to sulfadoxine-pyrimethamine. To our knowledge, this is the first study aiming to explore the combined effect of these factors on a cohort of pregnant women.Trial registration: This study has been registered on the ClinicalTrials.gov public website on 26th April, 2022. ClinicalTrials.gov Identifier: NCT05348746. Over the last twenty years, tremendous progress has been made on malaria control, averting an estimate of 1.5 billion cases and saving 7.6 million lives. However, despite the astonishing results achieved in the last two decades, COVID-19 pandemic added a crucial challenge to the fight against the disease. On one side, malaria control relies heavily on individual choice to seek care, and early messaging targeted on reducing SARS-CoV2 transmission advised people to stay home in case of fever. On the other side, supply chains that allow the delivery of malaria commodities, such as insecticide-treated nets or antimalarial drugs, have been disrupted, and healthcare workforce constrained. All these factors contributed to a reverse of the reducing trend in malaria cases and deaths that shifted, respectively, from 227 million and 534,000 in 2019 to 241 million and 602,000 in 2020 [According to the latest WHO World Malaria Report , Uganda Concerning the impact of COVID-19 on malaria care in Uganda, a study conducted by Namuganga et al. —except fP. falciparum infection and for development of severe malaria [In hyperendemic areas, pregnancy is a risk factor for both acquisition  of P. fa malaria . Younger malaria . Adverse malaria  and HIV  malaria .Apart from consistent use of ITN, malaria control in this population is based on two pillars: intermittent preventive treatment of asymptomatic women and appropriate management in case of illness . IPTp wiP. falciparum [In HIV negative women, a promising alternative to SP for IPTp is dihydroartemisinin-piperaquine , that showed to be more efficacious in reducing maternal malaria parasitemia and anemia at delivery, stillbirths and early infant mortality. In fact, SP efficacy may be decreased in areas with very high drug resistance and consistent presence of sextuple mutant haplotypes of lciparum . HoweverFor HIV positive patients, the current WHO guidelines recommend daily co-trimossazole prophylaxis.In most endemic countries, diagnosis heavily relies on the use of rapid diagnostic tests (RDT) which, however, are insufficiently sensitive in detecting the so-called subpatent infections—asymptomatic infections with low parasite densities—and infections due to parasites carrying the pfhrp2 and pfhrp3 gene deletions . These lP. falciparum malaria, WHO recommends 7 days of quinine + clindamycin. From the second trimester on, experience with artemisinin derivatives is increasingly reassuring: no adverse effects on the mother or foetus have been reported. Thus, treatment of uncomplicated P. falciparum malaria consists of three-day course of oral artemisinin-based combination therapy (ACT). On the other side, treatment of severe malaria does not differ from the one prescribed to non-pregnant women. Parenteral artesunate is the treatment of choice in all trimesters.Treatment of malaria differs according to gestational age. For women in their first trimester with uncomplicated kelch13) clearly showed the substantial difference of haplotypes between A675V isolates in Uganda and in Southeast Asia, which suggested that the mutation probably emerged independently in Africa and Southeast Asia. In Africa, potential factors that may contribute to a delayed emergence and spread of artemisinin resistance are the extent of acquired immunity, the rate of polyclonal infections and of chronic asymptomatic infections [Another substantial challenge for malaria case-management is resistance to first line drugs, namely artesunate and ACT. A recent paper published by Balikagala et al. documented, for the first time in African history, the presence of artemisinin resistance in a longitudinal study conducted in Gulu, Uganda . Accordifections . HoweverPfdhfr and Pfdhps mutations—conferring resistance to SP in P. falciparum—to be 89% for the quintuple mutated haplotype and 3.9% for the sextuple mutated haplotype, reaching 16.7% after one dose of SP. Today’s prevalence of the sextuple mutated haplotypes, potentially impairing the effectiveness of SP-IPTp is not known. Conceptual framework of the study is provided in Fig. Furthermore, a study conducted by Mbonye et al.  in 2015 This will be a facility-based, prospective observational study, using quantitative methods of data collection. Semi-structured questionnaires will be administered to collect the data. The study will be conducted on three separate populations, that is: “Cohort of pregnant women”, “Antimalarial resistance sub-population”, and “COVID-19 impact population” for which study methods are described separately.The data will be collected following a cohort of pregnant women presenting to antenatal care visits. We shall have both a retrospective cohort for the period January 2018 to December 2021 to determine the Impact of COVID-19 pandemic on malaria control and a prospective cohort for the period July 2022 to June 2024 to determine the incidence of malaria related adverse maternal and foetal outcomes. For the prospective cohort, recruitment will take place at ANC clinic. Collected data will explore the practices towards malaria prevention during the COVID-19 pandemic, malaria and COVID risk-perception and use of insecticide-treated nets, while and follow up will investigate access to antenatal care visits, administration of IPTp, healthcare seeking behaviour in case of fever. Follow-up will end at delivery, when maternal and foetal outcomes will be collected.P. falciparum malaria in the study sites. For individuals included in this population, clinical data will be collected, and blood samples to be sent to Italian National Institute of Health, Rome, for detection of genetic markers of resistance to artemisinin derivatives and sulfadoxine-pyrimethamine.To estimate the epidemiological burden of resistance to first-line drugs for treatment and prevention of malaria in pregnant women, a separate sub-population of women diagnosed with positive malaria parasitaemia will be recruited. Prevalence of antimalaria resistance will be evaluated with a cross-sectional analysis of genetic polymorphisms in plasmodium parasites isolated from blood samples collected for the period July 2022 to June 2024. Individuals eligible for this subpopulation will be all pregnant women presenting with microscopically confirmed To investigate the impact of COVID-19 pandemic on malaria care, we will conduct a retrospective time-interrupted series. Data will be collected at multiple and equally spaced time points (monthly) comparing trends in two different time periods: “pre-pandemic”, from January 2018 to December 2019, and “during pandemic”, from January 2020 to December 2021. Facility-based aggregate data will be extracted about the following indicators: total admissions in maternity ward, total deliveries in maternity unit, women presenting to first ANC contact, women presenting to fourth ANC contact, administration of at least one dose of IPTp, administration of at least three doses of IPTp, number of pregnant women presenting to outpatient visits, number of pregnant women diagnosed of malaria during outpatient clinic, number of pregnant women diagnosed of severe malaria, total number of stillbirths.Inclusion criteria:All pregnant women at any gestational age presenting to the study sites, both at the emergency department, outpatient or ANC clinic will be eligible to participate in this study. Inclusion criteria will be:Pregnancy confirmed by obstetrical visit, positive urine pregnancy test or intrauterine pregnancy by ultrasoundProvision of informed consentPlan to deliver in the hospitalOngoing labor or deliveryExclusion criteria:All pregnant women presenting to Aber Hospital and selected healthcare facilities with microbiologically confirmed malaria will be eligible for recruitment.Microscopically-confirmed diagnosis of malariaPregnancy confirmed by obstetrical visit, positive urine pregnancy test or intrauterine pregnancy by ultrasoundProvision of informed consentAgreement to avoid antimalarial medications given outside the Uganda Clinical Guidelines Inclusion criteria:Too ill to participate in the study according to clinician judgmentIntake of any antimalarial medication before recruitmentExclusion criteria:COVID-19 impact populationfacility-based census to include all pregnant mothers who sought care at the study sites for the period January 2018 to December 2021.Prospective çohort of pregnant womenFor the retrospective cohort investigating the impact of COVID-19, we shall use a Antimalarial resistance populationFor the prospective cohort, the sample size was estimated using the sample size estimation function in STATA12 for two-sample comparison of proportions. Null hypothesis: p1 = p2, (no difference in preterm birth rates), where p1 is the preterm birth rate among pregnant mothers diagnosed with malaria (exposed group) and p2 is the preterm birth rate among pregnant mothers with no diagnosis of malaria (non-exposed group). Assuming a type I error, alpha, of 0.050 for two-sided hypothesis, power of the study at 0.80, p1 = 0.075 and p2 = 0.039 based on the preterm birth rates reported in the Uganda Birth Cohort Study conducted from 2014–2016 in 12 districts in rural northern and southwestern Uganda [Given the total population of 779,600 in Oyam and Kole districts. Given that, based on Uganda bureau of statistics (UBOS), the total number of pregnant women in the two districts is expected to be 5% of the total population ; the expected rate of parasitemia among pregnant mother is expected to be 27%  ; the expAlready existent good laboratory capacity for diagnosis of malaria.Presence of an experienced microscopist.Antenatal care visit volumes of at least 50 visits per month.Good quality of routine service offered to patients with malaria as per periodic quality of care assessment.Sampling of health Units: The study will use purposive sampling to include Aber Hospital, Aboke HCIV and Atipe HCIII as the site for enrollment. This is based on the following criteria:Participants coming to the sampled in healthcare facilities that will meet the eligibility criteria will be included in the study. Consecutive enrollment of participants will be undertaken up to when the minimum sample size required for the study will be met.Thick and thin blood smears will be stained with 2% Giemsa and read by experienced laboratory technologists. Parasite densities will be calculated by counting the number of asexual parasites per 200 leukocytes , assuming a leukocyte count of 8000/µl. A blood smear will be considered negative when the examination of 100 high power fields does not reveal asexual parasites. Gametocytemia will also be determined from thick smears. Thin smears will be used for parasite species identification. At the time of delivery, recruited women will be screened for parasitemia on placental blood. This will be done by microscopy, with the same methods described above.The blood samples of the patients will be collected using filter paper (Whatman 3 MM) during admission to the healthcare facility. The dried blood spots (DBSs) will be collected through a finger prick (three drops of blood per participant) on filter papers which will be dried and kept in plastic bags with desiccant and stored in boxes in a cool dry place at room temperature before being transferred at the ISS for molecular diagnosis and drug resistance analysis.The collected blood samples will be shipped to Italy for advanced polymorphism analysis. During shipment, all the samples will be stored in a dry, cool place at room temperature to the Italian National Institute of Health . To enhance local capacity building, one laboratory person from Aber hospital will attend a two weeks exposure at the reference laboratory in Italy.Plasmodium species. The genus-specific PCR will be followed by Plasmodium species-specific PCR amplification. Amplicons from the second PCR will be separated by electrophoresis on a 2% agarose gel and stained with ethidium bromide for visualization using ultraviolet trans-illumination. The presence of parasitaemia will be confirmed when the expected band size corresponding to P. falciparum, P. vivax, P. malariae and or P. ovale will be identified.Total genomic DNA will be extracted from filter blots (3MM Whatman) using the PureLink Genomic DNA Kits-Invitrogen, according to the manufacturer’s recommendation. Parasite identification is based on nested PCR assay targeting the 18S rRNA gene . The 18SP. falciparum drug resistance genes: Pfk13 propeller, Pfdhfr and Pfdhps.Target Pfk13 gene will be performed by PCR amplifications and subsequent sequencing. Analysis of Pfdhfr gene at codons 51, 59, 108 and Pfdhps gene at codon positions 436, 437, 540, 581, 613 will be done by means of amplifications and subsequent Sanger sequencing. Commercial oligonucleotide primer pairs for Pfk13 will be obtained based on the published article by Taylor et al. [dhfr and dhps genes primer pairs will be obtained based on the published article by Menegon et al. [P. falciparum 3D7 strain) will be used as reference in the numbering of nucleotide and amino acid positions. Molecular studies will be performed only for research purposes and will have no impact on the clinical management of study patients.The polymorphisms analysis of the propeller domain of the r et al. , whereasn et al. . The obtHIV and Syphilis will be measured according to the Uganda National Guidelines Blood glucose will be measured by Glucometer “Accu-Chek Active”, Narang Medical LTD.Blood haemoglobin levels will be measured by Hemoglobin Testing System “Mission Ultra Hb”, Narang Medical LTD.For descriptive purposes continuous and ordinal variables data will be expressed as median with interquartile range. For categorical variables, percentages are calculated. Student’s t-test or analysis of variance (ANOVA) will be used to compare normally distributed numerical variables. Mann Whitney U-tests and Kruskal–Wallis tests will be used to compare numerical variables when normality cannot be assumed, while chi-squared tests will be used to compare categorical variables.Association analysis will be carried out to identify risk factors for Plasmodium infection and adverse maternal or foetal outcomes. We will compare behavioural factors and adherence to IPTp (and type of IPTp regimen) to the incidence of symptomatic/severe malaria and adverse neonatal or foetal outcomes . Multivariable logistic regression models will be used to identify independent risk factors for the same clinical outcomes. A forward and backwards stepwise approach will used to include variables into the models, with a limit of P < 0.2.A P-value of < 0.05 will be considered statistically significant. Final analyses will be conducted after the end of patient recruitment while interim analyses are planned at half 7 months from the incipit. Statistical analysis will be performed with R-software .Contact 1: Anytime ≤ 12 weeks.Contact 2: 13–20 weeks of gestation.Contact 3: 21–28 weeks of gestation.Contact 4: 30 weeks of gestation.Contact 5: 34 weeks of gestation.Contact 6: 36 weeks of gestation.Contact 7: 38 weeks of gestation.Contact 8: 40 weeks of gestation.According to the 2021 Essential Maternal and Newborn Clinical Care Guidelines for Uganda, for a woman with a normally progressing pregnancy the standard recommendation is a minimum of eight antenatal visits .Contact In addition, participants will be instructed to come to the clinic every time they are ill and will be evaluated at this point too. Outcomes will be assessed at the delivery or/and at the discharge if admitted to the hospital for any other causes related with the pregnancy or malaria Table .Table 1SData will be collected using semi-structured questioners and managed using REDCap electronic data capture tools hosted at “Catholic University of the Sacred Heart”, Rome, Italy , and wilPregnant women will be asked for written informed consent to participation to the study. In line with the Ugandan National Guidelines for Research involving Humans as Research Participants , women bWe shall administer consent at two levels; first we shall administer and obtain consent for participation in the study and publication of future results and thereafter consent for collection and transportation of blood samples to Italy. The blood samples collected will be used only for the purpose of this study and will not be stored beyond the current study project. All unprocessed blood samples will be destroyed from the department of infectious disease, Italian National Institute of Health as biological sanitary waste, at the end of the project. The same standard of care will be guaranteed to all individuals irrespectively of participation. The dignity of study participants will be guaranteed by the investigators, as well as data confidentiality and the right to withdraw data and/or biological samples at any time. Consent to pregnant women and to mature and emancipated minors will be obtained according to Uganda Human Subjects Protection Guidelines .For all malaria positive samples processed for molecular analysis in the present study, identifying information will be removed to provide appropriate protection of medical confidentiality and privacy. In this way, sensitive data cannot be linked or re-linked with identifiable human subjects, making anonymous each sample processed.Pfk13, Pfdhfr and Pfdhps genes will be performed by advanced amplifications techniques and subsequent Sanger sequencing in a highly specialized scientific laboratory. The blood samples collected in this study will be sent to a laboratory in the Italian National Institute of Health, Italy, in order to have access to the latest gene amplification techniques. To enhance local capacity building, one laboratory staff from Aber Hospital will be supported to attend a two weeks exposure at the reference laboratory in Italy.The polymorphisms analysis of the propeller domain of the The present study has been approved by the Lacor Hospital Institutional Research and Ethics Committee (prot. no LACOR-2022-95).Results from this research will be disseminated at various fora, including local and international scientific conferences, the Ministry of Health, technical working groups and meetings at district offices in Lango region involving participation of local leaders . To better reach enrolled communities, the findings of the study will also be disseminated in places of worship and community health service points using the community outreaches being organized by CUAMM and the district local governments in Oyam and Kole districts. Findings will also be published in a peer reviewed journal.


Plasmodium parasites and 170 healthy controls without a history of malaria. Genotyping of the β-like globin C-158T, G79A, C16G, and C-551T SNPs was performed using a polymerase chain reaction-restriction fragment length polymorphism approach. Risk association, linkage disequilibrium (LD), and haplotype analyses of these SNPs were assessed. This study found that the variant allele in the C-158T and C16G SNPs were protective against malaria infections by 0.5-fold, while the variant allele in the G79A SNP had a 6-fold increased risk of malaria infection. No SNP combination was in perfect LD, but several haplotypes  were identified to link with different correlation levels of malaria risk in the population. In conclusion, the C-158T, G79A, and C16G SNPs in the β-like globin gene are associated with the risk of malaria. The haplotypes  identified in this study could serve as biomarkers to estimate malaria risk in the population. This study provides essential data for the design of malaria control and management strategies.Single nucleotide polymorphisms (SNPs) in the β-like globin gene of the human hosts to the risk of malaria are unclear. Therefore, this study investigates these associations in the Sabah population, with a high incidence of malaria cases. In brief, DNA was extracted from 188 post-diagnostic blood samples infected with  Plasmodium parasites transmitted by the Anopheles mosquitoes’ bites. Five Plasmodium species have been identified to infect humans, including Plasmodium falciparum, Plasmodium vivax, Plasmodium ovale, Plasmodium malariae, and Plasmodium knowlesi. In 2020, malaria cases were estimated to increase to 241 million globally [Malaria is a zoonotic disease caused by Plasmodium parasites, and about 10,000 of them eventually live in active foci areas [P. knowlesi is the most commonly reported species in Sabah. Many studies have been conducted to understand better the effects of this Plasmodium species in Sabah, including risk factors for infection with P. knowlesi and the genetic diversity of P. knowlesi [Malaysia is also one of the countries significantly impacted by the malaria disease. Approximately 1.3 million of the Malaysian population are at risk of being infected by the knowlesi ,5,6.Variants in a human gene are regularly associated with the risk of certain diseases. For example, the C-158T rs7482144) single nucleotide polymorphism (SNP) in the 5′ region of the hemoglobin subunit gamma 2 gene is linked with higher fetal hemoglobin levels, sickle cell anemia, and thalassemia 482144 si. FurtherSabah, a state with many rural areas and a hotspot for malaria transmissions, reported the highest malaria incidences from 2013 to 2017 compared to Sarawak and Peninsular Malaysia [Plasmodium parasites were collected from the Public Health Laboratory, Kota Kinabalu, Sabah. Among these samples, 134 blood samples were infected with P. knowlesi, 28 were infected with P. falciparum, and 26 were infected with P. vivax, as molecularly validated previously using the PlasmoNexTM multiplex assay [Plasmodium parasite, and (ii) the controls were not living within the Kota Kinabalu areas and with a history of malaria infection. The genomic DNA was extracted from these blood samples using a previously described method [A total of 188 post-diagnostic blood samples infected with ex assay . Besidesd method . The stuGoTaq® Flexi Buffer , 1 unit of GoTaq® Flexi DNA polymerase , 1.5 mM of MgCl2 solution, 0.2 mM of dNTP mixture, and 0.2 µM of both forward and reverse primers (2O) until a final volume of 20 µL. The PCR conditions were set as follows: initial denaturation at 94 °C for 4 min; amplification of 35 cycles at 94 °C for 30 s, annealing temperature according to different primer sets (A polymerase chain reaction (PCR) master mix consisting of 100 ng of DNA template, 1X of  primers  was prepmer sets  for 30 sTM Buffer  was prepared. The mixture was topped-up with sdH2O to a final volume of 15 µL and incubated overnight at 37 °C. The digested products were analyzed in 2–3% agarose gel stained with ethidium bromide  according to the respective SNPs , and 1X  bromide , and thep-value less than 0.05 was considered statistically significant.The odds ratio (ORs) and 95% confidence interval (CI) were calculated using the SPSS software ver. 22.0 . The LD and haplotype analyses of the SNPs were determined using the SHEsis software . A p-valp = 0.027) and a lower risk of infection by P. knowlesi     . On the = 0.026) . Subject= 0.017) . There w= 0.017) .2 value (r2 = 0.001) (2 (<50%) values.The LD analysis showed that the C-158T and G79A SNPs combination was in a complete LD (D’ = 1.000) but with an extremely low r= 0.001) , indicatp < 0.001), whereas the haplotypes CGCT  and CGGC  were protective against the risk of malaria infection. Although the haplotypes CGGT and TGGT appeared to increase the risk of malaria infection, they were not statistically significant (p > 0.05).Plasmodium species can provide useful information for public health officials to design interventions for malaria cases, aligning with the vision of World Health Organization and the global malaria community in creating a world free of malaria. Several SNPs in the β-like globin gene have been reported to have different malaria susceptibility effects. This study investigated the association of the β-like globin C-158T, G79A, C16G, and C-551T SNPs with the risk of malaria in the Sabah population of Malaysian Borneo.Sabah, Malaysian Borneo, had the highest malaria cases and deaths among all states in Malaysia from 2013 to 2017 [P. knowlesi infection. Similarly, a pediatric malaria study reported that the spread of the Plasmodium parasite was retarded by HbF-containing red blood cells [The C-158T SNP is located upstream of the γ-globin gene, which directly affects the production of hemoglobin F (HbF) . This isod cells . This suP. falciparum infection. Interestingly, a previous study has empirically shown that the variant allele created an alternative splicing site, which led to the production of a mutated beta-chain and is associated with a blood disorder, the HbE beta-thalassemia [Plasmodium infection [Plasmodium infection protection should be further explored.Besides that, the G79A SNP is a mutation at codon 26 of the β-globin gene, which causes the hemoglobin E (HbE) variant . This stlassemia . Despitenfection . As a reP. vivax infection by 0.5-fold, as observed in the present study. However, a recent study reported that infants with lower hemoglobin levels are not protected against malaria infection in a Papua New Guinean population [The C16G SNP is located in the splicing region of the β-globin gene, and in silico studies revealed that the variant G allele could contain two possible branch sites that might induce a truncated protein ,29, resupulation . Since tPlasmodium species and Anopheles mosquitoes, have to be mutually considered.This study did not observe a significant association between the C-551T polymorphism and malaria risk. Despite its insignificant association, a correlation has been observed between the genotype distribution and malaria incidence. For instance, the Indian population has the highest C-551T polymorphism (79%), whereas the Greece population has the lowest C-551T polymorphism (37%) . SimultaP. vivax isolates were associated with different malaria transmission rates [A recent genome-wide association study on malaria parasites reported that LD and large haplotype blocks from 3.6 kb to 14.0 kb at chromosome 8 of on rates . TherefoOne of the limitations of this study is that the present study only focuses on the genetic aspect, and the hemoglobin levels of the human hosts were not assessed. Therefore, the interplay between the SNPs in the β-like globin gene and hemoglobin levels to the risk of malaria infection could not be estimated. Future studies should consider this limitation in their study designs.In conclusion, this is the first study to investigate the association of the β-like globin SNPs with the risk of malaria in the Sabah population of Malaysian Borneo. This study suggests that the C-158T and C16G SNPs are protective against malaria infection, while the G79A SNP is an increased risk factor for malaria infection. Several haplotypes  with different correlation levels to malaria risk were identified, and they could serve as biomarkers for malaria risk estimation in the population. The data of this study could be essential to understanding the hosts’ immune responses to malaria susceptibility and the development of antimalarial drugs to treat malaria.


Asymptomatic carriage of malaria parasites is common in high transmission intensity areas and confounds clinical case definitions for research studies. This is important for investigations that aim to identify immune correlates of protection from clinical malaria. The proportion of fevers attributable to malaria parasites is widely used to define different thresholds of parasite density associated with febrile episodes. The varying intensity of malaria transmission was investigated to check whether it had a significant impact on the parasite density thresholds. The same dataset was used to explore an alternative statistical approach, using the probability of developing fevers as a choice over threshold cut-offs. The former has been reported to increase predictive power.Data from children monitored longitudinally between 2005 and 2017 from Junju and Chonyi in Kilifi, Kenya were used. Performance comparison of Bayesian-latent class and logistic power models in estimating malaria attributable fractions and probabilities of having fever given a parasite density with changing malaria transmission intensity was done using Junju cohort. Zero-inflated beta regressions were used to assess the impact of using probabilities to evaluate anti-merozoite antibodies as correlates of protection, compared with multilevel binary regression using data from Chonyi and Junju.Malaria transmission intensity declined from over 49% to 5% between 2006 and 2017, respectively. During this period, malaria attributable fraction varied between 27–59% using logistic regression compared to 10–36% with the Bayesian latent class approach. Both models estimated similar patterns of fevers attributable to malaria with changing transmission intensities. The Bayesian latent class model performed well in estimating the probabilities of having fever, while the latter was efficient in determining the parasite density threshold. However, compared to the logistic power model, the Bayesian algorithm yielded lower estimates for both attributable fractions and probabilities of fever. In modelling the association of merozoite antibodies and clinical malaria, both approaches resulted in comparable estimates, but the utilization of probabilities had a better statistical fit.Malaria attributable fractions, varied with an overall decline in the malaria transmission intensity in this setting but did not significantly impact the outcomes of analyses aimed at identifying immune correlates of protection. These data confirm the statistical advantage of using probabilities over binary data.The online version contains supplementary material available at 10.1186/s12936-022-04346-9. Asymptomatic carriage of malaria parasites is highly prevalent in areas with high malaria transmission as a result of naturally acquired immunity . It is, To overcome this problem of misdiagnosis, different studies have based the case definition of febrile malaria with parasite density above a locally defined threshold. The computation of malaria attributable fractions (MAF) or the proportion of fevers due to malaria parasites has been used to define different thresholds for parasitaemia , 3.The classical method for deriving the attributable fraction is a simple numerator denominator approach  which isMalaria transmission intensity has been found to strongly influence the attributable fractions. In a study conducted in two areas with different transmission intensities in Kilifi at the coast of Kenya, a MAF of 50.2% was estimated for Ngerenya, the low transmission site and 47.9% for Chonyi, the high transmission site. In the study, the logistic regression method was applied and derived a parasite density threshold of 2500 parasites/an coast –13.Significant reductions in malaria transmission and admissions have been reported over the last decade in endemic countries in Africa  and in pThe probability of fever as a function of parasite density and the optimal parasite thresholds was estimated using logistic regression , 17. TheSeveral articles –24 have This research utilized cohort data from Junju and Chonyi sub-counties in Kilifi County, which is part of the Kilifi Health and Demographic Surveillance System (KHDSS) on the coastal region of Kenya Fig. A cross-sectional bleed survey was done every year at the beginning of the malaria season (March–May) for the Junju cohort as shown in Table For the parasitaemia determination by microscopy, the number of asexual-stage parasites/200 leukocytes was counted, and parasitaemia was estimated based on actual or assumed  leukocyte count measured for each blood smear.A comparison of logistic regression and Bayesian latent class models as estimators of malaria positivity was done. For both approaches, the relationship between the risk of fever and parasite density was carried out separately for each year and age group. A parasite density cut-off was estimated from the logistic approach and probabilities of children with different levels of parasitaemia were estimated from the Bayesian approach using R  and OpenA logistic regression model was fit to the data, modelling the risk of fever as a continuous function of the parasite density. The model was of the form mentclass2pt{minimFor the Bayesian latent class model, the parasite density was resolved to a mixture of two multinomial distributions. One component The parameters To estimate the probability, Sensitivity and specificity of various cut-off values for parasite density were estimated by Multi-level logistic and zero-inflated models were used to investigate the association between high versus low merozoite antibodies and clinical malaria. Various antibody concentrations were applied as cutoffs to define the high and low responders . The resA total of 268 participants from Chonyi and 4722 participants from Junju, Kilifi County were recruited in 2000 and from 2005 to 2017 respectively. Approximately 300 or more participants were followed up each year with average recruitment age of 6.5 years (ranging between 1 month old to 16 years) as shown in Table Plasmodium falciparum was also artificially high during this period since the participants were recruited during the malaria season. A decline in the prevalence of P. falciparum parasite was observed between 2006 to 2013 from 30.21% to 8.78%. This was followed by a slight increase in 2014 and 2015 then another decline in 2016 to 4.32% in 2017.Table The probability that a fever case was malaria attributable at a given parasite density entclass1pt{minimaAn interval estimate for the prevalence of malaria fever was estimated using the Bayesian latent class model. The individual probabilities from the Bayesian fit for non-febrile participants with parasitaemia were adjusted using the interval estimate for the prevalence of malaria fever. This is shown in Additional file The MAF estimates were higher for older age groups than the children < 1 year as shown in Fig. P. falciparum merozoite antigens for a study done in Kilifi was used. Specifically, the data had antibody measurements for the survey conducted in Junju in the year 2008 and a subset of the Chonyi cohort in the year 2000 [Predicted probabilities from Bayesian latent class model were compared with the binary outcome defined using logistic parasite density thresholds in identifying correlates of disease protection. To compare the perfomance, data on antibody responses to selected ear 2000 .A cut-off of 2500 parasites/µl Additional file In areas with high malaria transmission, differences in the prevalence of malaria fever can occur due to change in transmission intensities or differences in levels of immunity in various subsets of the population like age groups . The preTherefore, it is important to review MAF and case definitions with changing transmission settings. Furthermore, transmission intensity correlates with the rate of acquisition of natural immunity . A decreA strong rationale for developing malaria vaccines comes from cohort studies, which show that individuals continuously exposed to malaria develop immunity that initially prevents death from severe disease, and subsequently recurrent illness , 29. TheThe logistic power model is the widely used technique for case definition , 7–9, 30Continuous variables, like the probabilities used here have been shown to have more variation information and statistical power and are sometimes preferred over the categorization of data . SeveralFor this study bi-weekly active surveillance was conducted. Therefore, short-lived asymptomatic infections below the level of detection by microscopy and exposure that does not result in a blood stage infection may have been missed. Parasite density cut-offs plus fever are used mostly in malaria endemic studies to inform policy. This study examined whether the varying intensity of malaria transmission affected the estimation of optimal cutoffs using the Junju cohort. Varying thresholds estimates were observed; however, this did not have a substantial impact on the number of febrile malaria individuals in this study. This research demonstrates the statistical advantage of utilizing probability outcomes over parasite thresholds. It has been shown that continuous variables, like the probabilities used, have more variation information and statistical power. Sometimes this is preferred over the categorization of data, however, a training dataset is required to estimate the probabilities .In the malaria attributable fraction estimation, this study assumed independence of the malaria episodes of individuals with repeated measurements over the years. This was a major limitation; however, this was handled by considering the first 6 months of follow-up for the study participants per year of recruitment to reduce inter-dependence.The present study compares the performance of the logistic and Bayesian models in estimating MAF. Utilization of probabilities estimated from the Bayesian estimator has a better statistical fit in modelling the association of correlates of disease compared to the dichotomization approach of cases and controls using parasite thresholds from the logistic estimator. Another objective was to investigate whether the varying intensity of malaria transmission had a significant impact on parasite density thresholds.Results from Junju and Chonyi cohorts verify the validity of using the probability outcome to identify correlates of disease protection while still having a better statistical fit. The computational time to fit the zero-inflated models was higher compared to the binary-based regression models and a training class is required for the latent class models which can be a limitation for some cohort designs.Approaches to estimating an individual’s marginal probabilities of clinical malaria over a given follow-up time would be of importance for creating parsimonious models. Further studies to compare the probabilities estimated from models utilizing the quantitative nature of the parasite densities without grouping the data in conjunction with changing transmission would be valuable.Additional file 1: Table S1. Parasites/µL cut off using Logistic regression. Fig S1. Distribution of predicted probabilities. Fig S2. Comparison of probabilities for Bayesian and Logistic, Junju 2008. Table S2. Anova test for Figure 2B. Fig S3. Comparison of probability of febrile and non-febrile. Fig S4. Predicted probabilities over age groups. Fig S5. Sensitivity and specificity of the different years. Fig S6. Posterior estimates of AF.