IFNL3/IFNL4 genomic region, which encodes type III interferons, have been strongly associated with clearance of hepatitis C virus. We hypothesized that type III interferons might be important for the immune response to other pathogens as well. In a cohort of 914 Malian children, we genotyped functional variants IFNL4-rs368234815, IFNL4-rs117648444, and IFNL3-rs4803217 and analyzed episodes of malaria, gastrointestinal, and respiratory infections recorded at 30,626 clinic visits from birth up to 5 years of age. Compared to children with the rs368234815-TT/TT genotype (IFN-λ4-Null), rs368234815-dG allele was most strongly associated with an earlier time-to-first episode of gastrointestinal infections (p = 0.003). The risk of experiencing an infection episode during the follow-up was also significantly increased with rs368234815-dG allele, with OR = 1.53, 95%CI (1.13–2.07), p = 0.005 for gastrointestinal infections and OR = 1.30, 95%CI (1.02–1.65), p = 0.033 for malaria. All the associations for the moderately linked rs4803217 (r2 = 0.78 in this set) were weaker and lost significance after adjusting for rs368234815. We also analyzed all outcomes in relation to IFN-λ4-P70S groups. Our results implicate IFN-λ4 and not IFN-λ3 as the primary functional cause of genetic associations with increased overall risk and younger age at first clinical episodes but not with recurrence or intensity of several common pediatric infections.Genetic polymorphisms within the  Despite the tremendous progress, pediatric mortality remains unacceptably high. In 2015, of 5.9 million deaths before the age of five that occurred globally, 30% of deaths were caused by pneumonia, malaria, and diarrhea [For example, variable ability to clear hepatitis C virus (HCV) infection is associated with polymorphisms in the genetic locus on chromosome 19 that encodes the family of type III interferons (IFNs) –6. TheseHumans have four type III IFNs, all encoded within the 70 kb region on chromosome 19. Three of these IFNs (IFN-λ1-3) can be inducibly generated in all the individuals, while IFN-λ4 is produced only in about 50% of the world population, including 90% of individuals of African ancestry, versus only 50% of Europeans and 10% of Asians , 9. The IFNL4 gene. The open reading frame for IFN-λ4 protein is created by the dG allele, whereas the TT allele results in the truncated, non-functional protein (IFN-λ4-Null) [IFNL4 SNPs (rs368234815 and rs117648444) stratifies all individuals in groups of those not producing any IFN-λ4 protein, producing only weak IFN-λ4-S70 or strong IFNλ4-P70 protein. The ability to produce strong IFNλ4-P70 protein was associated with impaired HCV clearance [The production of IFN-λ4 is controlled by a frameshift dinucleotide polymorphism, rs368234815-dG, within the first exon of the λ4-Null) . The comlearance , indicatIFNL3/IFNL4 region may also demonstrate significant associations because of population-dependent moderate to high linkage disequilibrium in this genomic region. One of these linked variants is a single nucleotide polymorphism (SNP) rs4803217, which is located within the 3′UTR of IFNL3; this variant was proposed to be functional and affecting IFN-λ3 protein by modulating the stability of IFNL3 mRNA [IL28B (IFNL3) marker, is located within the first intron of IFNL4 and often used as a proxy for the functional rs368234815 but without any known functional effect.Although rs368234815-dG is considered the causal variant due to its direct functional effect on the production of IFN-λ4 protein, whose biological activity might be modified by rs117648444 (P70S), additional polymorphisms in the NL3 mRNA . AnotherIFNL3/IFNL4 genetic variants and HCV clearance have been well established and replicated by multiple studies [IFNL4 genetic polymorphisms (rs12979860 and rs368234815) have been associated with the clearance of respiratory RNA viruses in children in Rwanda [IFNL4-rs368234815, IFNL4-rs117648444, and IFNL3-rs4803217. We analyzed clinical data collected during 30,626 routine and walk-in clinic visits from birth to up to 5 years of age. Specifically, we analyzed episodes of infections classified as malaria , gastrointestinal, and respiratory infections and evaluated their occurrence during follow-up time (ever/never), time-to-first episode, and the count of independent episodes. Our results provide new clues on the role of type III IFNs, and IFN-λ4, specifically, in genetic control of the immune response to several common infections in African children.The strong associations between the  studies –6. Beyonn Rwanda . To furtThe study used data and samples collected by the longitudinal birth cohort of 914 children enrolled between 2010 and 2016 in Ouelessebougou Community Health Center (CESCOM), located 80 km south of Bamako, Mali. At their antenatal visits, pregnant women aged 15–45 years were invited to participate in the study. The eligibility criteria included an absence of a chronic, debilitating illness and residence in Ouelessebougou district for at least 12 months before enrollment. The newborn children of the participating mothers were enrolled at delivery, and cord blood was collected. The study site is located in an area of intense but highly seasonal malaria transmission. The follow-up protocol included visits to the clinic scheduled monthly during the high malaria transmission season (June–December) and every two months during the dry season (January–May), as well as ad hoc walk-in clinic visits for any acute symptoms that could not wait for the next regularly scheduled follow-up visit. Clinical information was collected during all visits by project clinicians and recorded using standardized forms.P. falciparum malaria parasite detected by thick blood smear microscopy, with or without clinical symptoms. Clinical malaria was defined as fever >37.5 °C, presence of malaria-related symptoms, and either a positive Rapid Diagnostic Test for malaria antigens or visualized malaria parasites on thick blood smear microscopy. Severe malaria was defined as P. falciparum parasitemia and at least one of the following World Health Organization criteria for severe malaria—2 or more convulsions in the past 24 h, prostration (inability to sit unaided or in younger infants inability to move/feed), hemoglobin <5 g/dl, respiratory distress , or coma (Blantyre score <3). The presence of severe malaria symptoms but with negative tests for malaria antigens or parasites was attributed to other diseases. All other parasitemia episodes, including those that were asymptomatic, were classified as non-severe malaria. For ever/never analyses, the non-severe malaria group included children with malaria history who never had a severe malaria episode, while the severe malaria group included children with at least one severe malaria episode, regardless of the number of non-severe malaria episodes.Since the primary goal of this birth cohort was to explore host and parasite factors that influence susceptibility to malaria infection and disease during pregnancy and early childhood, detailed malaria-related symptoms were recorded at each visit. Per study protocol, malaria infection was defined as positivity for Standardized clinical forms also captured other symptoms, such as diarrhea and cough, and other signs of respiratory distress . Using a syndromic approach, we attributed diarrhea to gastrointestinal infections and cough/respiratory distress to respiratory infections. Although both gastrointestinal and respiratory infections could be caused by bacterial, viral, parasitic, or fungal infections, this information was not collected by the study, and a more detailed etiology could not be explored. All children received treatment based on their symptoms. Episodes of the same infection were considered independent if they occurred more than 28 days apart.Before enrollment, written informed consent was obtained from all study participants after receiving written and oral study explanations from a study clinician in their native language; the parents/guardians provided consent on behalf of their children. All methods were carried out in accordance with relevant guidelines and regulations. The study procedures were ethically approved by the Institutional Review Board of the National Institute of Allergy and Infectious Diseases (NIAID) at the US National Institutes of Health , and the Ethics Committee of the Faculty of Medicine, Pharmacy and Dentistry at the University of Bamako, Mali.Genomic DNA was extracted from cord blood clots using the QIAsymphony automation system with protocol Blood_200_V7_DSP and the DSP DNA Mini Kit (Qiagen) by the Cancer Genome Research Laboratory of the DCEG/NCI. The DNA samples were tested by microsatellite fingerprinting  to confirm gender concordance with study records and exclude any mixed-up or contaminated samples.IFNL4-rs368234815, IFNL4-rs117648444, and IFNL3-rs4803217 by custom TaqMan genotyping assays as previously described [HBB polymorphisms rs334-T/A, HBB-Glu7Val  and rs33930165, HBB-Glu7Lys  were genotyped by Sanger sequencing as previously described [IFNL3 and IFNL4 SNPs did not deviate from Hardy–Weinberg equilibrium (HWE). All genotyping and sequencing were done in the Laboratory of Translational Genomics  blinded to demographic and clinical data. Merging with phenotypes and outcomes was done externally (NIAID) after all the genotyping was complete. Of the initial cord blood clots from 993 children, 21 (2.1%) were excluded due to poor quantity/quality of the extracted DNA. Of the remaining 972 samples, 38 samples (3.9%) were excluded due to discordant HBB status between the study records (determined based on gel electrophoresis) and DNA genotyping, 18 samples (1.9%) were excluded due to sex discordance between study records and Identifiler profiles and two samples (0.2%)—due to failed rs368234815 genotyping.DNA samples were genotyped for IFNL4-rs368234815 and IFNL3-rs4803217, which were evaluated assuming the additive and genotypic genetic models, using the TT/TT genotype or TT allele (IFN-λ4-Null) for rs368234815 and G/G genotype or G allele for rs4803217 as reference groups. Haplotypes and linkage disequilibrium metrics (D′ and r2) between the markers were analyzed and plotted using Haploview 4.2 [For each infection outcome, we considered the occurrence of at least one episode during the follow-up time (ever/never), time-to-first episode, and the count of independent episodes. The main outcome predictors were genetic variants HBB status coded as 0, 1, and 2 for AA (wild-type) vs. sickle-cell trait (A/S or A/C) vs. sickle-cell disease  was used as a covariate for all malaria-related analyses; sex and birth month  were tested but not significantly associated with any outcomes. Logistic regression models were used to evaluate the associations of IFNL4-rs368234815 with infections of interest, adjusting for the occurrence of other infections (ever/never) to examine whether these associations were specific to particular infection types.The occurrence of infection episodes (never/ever) was analyzed for both polymorphisms separately and in the same model using logistic regression models and adjusting for the follow-up time (weeks). Based on rs368234815 and rs117648444, all children were stratified into four groups: those not producing any IFN-λ4 protein (Null), producing only weak IFN-λ4-S70 protein, and one or two copies of the strong IFN-λ4-P70 protein. Time-to-first episode of each infection was analyzed with Kaplan-Meyer plots and Cox proportional hazards regression models. The number of infection episodes during follow-up was analyzed using Poisson models with overdispersion (Quasi-likelihood), adjusting for the length of follow-up as an offset in the model (offset = log(LastVisitAge)).https://cran.r-project.org/). The results were not adjusted for multiple testing.Since the study did not have permission to perform genome-wide analyses of genetic variants that could be used to adjust for possible population stratification, we used self-reported paternal tribe information as a proxy. Of the 21 tribes, four tribes accounted for 92.5% of all the children: Bamana—72.3%, Peulh/Fulani—9.2%, Soninke—6.0%, and Malinke—5.0%. The reported paternal tribe  was used as a random effect variable for logistic regression analyses (glmer package), Cox regression analyses with gamma frailty, and the analyses of infection episode counts per year (glmmPQL package). All the analyses were performed using specified packages in R , while one and two infection types were reported at 9407 (30.7%) and 1380 (4.5%) visits, respectively. Only 31 visits (0.1%) reported all three infection types at the same visit.Episodes of any infection  were reported at 12.7% of the routine and 76.3% of the walk-in visits , while malaria and gastrointestinal infection was the least common combination, with 0.8% of routine and 1.6% of walk-in visits , and IFNL3-rs4803217 alleles (T-66.7% and G-33.3%) were in moderate linkage disequilibrium (D’ = 0.98 and r2 = 0.78), IFNL4-rs117648444 alleles were present at A-10% and G-90% frequencies  in 18.6%, and homozygous variant genotypes associated with sickle cell disease –  in 0.9% of the children.The wild-type HBB, IFNL3 and IFNL4 polymorphisms were comparable to those in the populations of African ancestry in the 1000 Genomes Project, especially to the populations from West-Africa  and present the results adjusted for the tribes; unadjusted results are also presented in corresponding tables/figures.p = 0.005), malaria , p = 0.033), non-severe malaria , p = 0.047), but not of respiratory infections  , IFNL4-rs368234815 and time-to-first episode of each infection, which represents the earliest occurrence of symptoms of sufficient severity to require a visit to the clinic. The most significant association was observed for the IFNL4-rs368234815 and time-to-first episode of gastrointestinal infections  , but not for malaria overall and severe malaria  as those not producing any IFN-λ4 protein (Null), producing only weak IFN-λ4-S70 protein or producing at least one copy of the strong IFN-λ4-P70 protein . Becausep = 0.009, Table Table We leveraged extensive follow-up data from a large and well-characterized birth cohort from Mali with genetic analysis of three polymorphisms functionally relevant for two type III IFNs, IFN-λ3 and IFN-λ4. We showed that, compared to IFN-λ4-Null children, children with genetic ability to produce IFN-λ4, and especially those producing functionally strong IFN-λ4-P70 protein, were more likely to experience gastrointestinal infections, manifested by diarrhea, with the first infection episode occurring at a younger age. Although weaker, we also observed a similar trend for malaria and respiratory infections.While using diarrhea and cough/respiratory distress as proxies for gastrointestinal and respiratory infections limits the etiological inferences on the observed genetic associations, these patterns are informative when considering the most common causes of these manifestations. For example, in young children, acute diarrhea with or without vomiting is likely to be of viral etiology, caused by infection with rotavirus (RoV), norovirus (NoV) or astrovirus (AstV) , 19, or Because human enteric viruses are difficult to study in vitro , 25, mosIFNL4-rs368234815-dG allele might indicate an impaired immune response in these children due to the production of IFN-λ4. However, if diarrhea can be considered as a mechanism to purge those pathogens from the gastrointestinal tract, these results might also indicate an enhanced innate immune response, as has been suggested based on mouse studies [The higher risk and earlier episodes of gastrointestinal infections in children with  studies . The magSimilarly, we broadly defined respiratory infections using cough/respiratory distress manifestations regardless of their etiology. Respiratory infections cause an estimated 1.9 million deaths annually in children under five, with 70% of these deaths occurring in Africa and Southeast Asia . Some ofIFNL4-rs12979860-T and rs368234815-dG alleles with impaired clearance of respiratory RNA viruses in nasal swabs of children from Rwanda [p = 0.045) with an earlier occurrence of respiratory infections in children with IFNL4-rs368234815-dG allele, but not reaching statistical significance. This could be because respiratory symptoms recorded during clinic visits in our study were defined very broadly, were very common (experienced by 97.7% of children during follow-up), and occurred very early in life, with the median timing of the first respiratory episode being at 16 weeks of age.A genetic association was reported for m Rwanda . In our Although type III IFNs have been recognized for their antiviral role, their involvement in bacterial and fungal infections has also been reported . Our finIFNL3 mRNA and possibly accounting for the association in the IFNL3/IFNL4 region [IFNL4-rs368234815 as a primary associated and functional genetic variant in this region. The significant linear trends for associations between the inferred biological activity of IFN-λ4 (as Null-weak-strong IFN-λ4 protein) and infection episodes provide additional support to our hypothesis that IFN-λ4 activity increases the risk of acute clinical GII infections and malaria. We did not observe significant differences in the counts of infection episodes in children with various IFNL4 genotype or IFN-λ4-P70S groups. Thus, the biological activity of IFN-λ4 is likely to be associated with increased overall risk and younger age at first clinical episodes but not with recurrence or intensity of infections.While rs4803217 was previously suggested as a functional variant affecting the stability of 4 region , this SNThe strength of our study includes its large sample size, longitudinal design, and detailed follow-up data collected over 30,626 clinic visits over almost 5 years that captured most, if not all, episodes of common infections in these children. The limitations of our study include using a syndromic approach of utilizing proxies for infections, such as diarrhea for gastrointestinal infections and cough/respiratory distress for respiratory infections, even though these infections could be caused by multiple factors . Due to IFNL4 genotype  suggests that response to other clinically relevant RNA viruses, including SARS-CoV-2 that causes COVID-19, might also be impaired in carriers of this genotype, and some support for this idea has recently been reported [Clearance of several RNA viruses associated with reported .In conclusion, our study suggests that IFN-λ4 negatively affects the immune response to several infections in young children in Africa, in line with the overall unfavorable role of IFN-λ4 in various health conditions , 41. OurSupplemental Material


Plasmodium falciparum is responsible for the vast majority of (severe) clinical malaria cases in most African settings. Other Plasmodium species often go undiagnosed but may still have clinical consequences.Plasmodium malariae infections from Eastern Uganda (aged 2–39 years) are presented. These infections were all initially mistaken for P. falciparum, but Plasmodium schizonts (up to 2080/µL) were identified by microscopy. Clinical signs included history of fever and mild anaemia.Here, five cases of P. falciparum transmission, where rapid diagnostic tests that detect only P. falciparum antigens are commonly used, non-falciparum malaria cases may be missed.These findings highlight the importance of considering non-falciparum species as the cause of clinical malaria. In areas of intense  Plasmodium falciparum. Thus, it is not surprising that P. falciparum remains the focus of malaria control programmes and most malaria research in Africa. Other Plasmodium species are endemic in sub-Saharan Africa, including Plasmodium ovale, Plasmodium vivax and Plasmodium malariae [P. malariae may last for years, and can cause severe complications in approximately 3% of cases, including nephrotic syndrome, splenomegaly and anaemia [P. malariae infections have been observed following treatment of P. falciparum infections with artemisinin-based combination therapy (ACT) [In 2019, an estimated 229 million cases and 409,000 deaths due to malaria were reported globally [py (ACT) , 6.Plasmodium species can be differentiated by microscopy but this requires laboratory infrastructure and technical expertise. Infections with P. malariae are typically characterized by low parasitaemia [Plasmodium malariae is often missed or misdiagnosed by microscopy [P. malariae infection, either alone or mixed with other species, in population surveys in African countries, where prevalence of up to 12% has been reported [Different sitaemia  and the sitaemia . Plasmodcroscopy , 10. Howreported , 12.P. falciparum; use of these HRP2 RDTs is likely to contribute further to under-diagnosis of P. malariae infections.Rapid diagnostic tests (RDTs), which are simple to use and require minimal training, are increasingly being used to diagnose malaria. In Africa, most RDTs detect histidine-rich protein 2 (HRP2), an antigen that is specific for P. malariae. These cases were coming from an area of intense P. falciparum transmission in Eastern Uganda and were initially misdiagnosed as P. falciparum by microscopy.Here, five cases are presented of symptomatic, uncomplicated P. falciparum is perennial and intense [Patients described in this case report presented at either Masafu General Hospital or Nagongera Health Centre IV. Masafu General Hospital is a district referral facility in Busia District, an area in south-eastern Uganda that borders Kenya and Lake Victoria, where transmission of  intense . Nagonge intense . Between intense .P. falciparum, clinic technicians all received formal microscopy training and thick and thin blood films were used for malaria diagnosis, instead of RDTs. Haemoglobin measurements were performed by Hemocue™ .In both settings, hospital and health centre, malaria diagnosis is routinely performed by HRP-2 based rapid diagnostic test . However, as part of a research study on On January 30th 2020, a 15-year-old male presented to the OPD with history of fever, headache and joint pain for 3 days with no evidence of severe malaria or danger signs. At initial presentation, he had an axillary temperature of 36.8 °C and his physical examination was within normal limits. Malaria parasites were reported on the thick and thin blood film. By thick blood smear, total parasite count was 896 parasites/μL; malaria schizonts were observed at 176 parasites/μL . Haemoglobin concentration was 12.3 g/dL. The patient was diagnosed with P. falciparum malaria.P. falciparum malaria.On February 19th 2020, a 20-year-old female presented to the OPD with history of fever for 3 days and headache, weakness and joint pain for 2 days. Her temperature on admission was 37.2 °C and her physical examination did not show abnormalities. After reading the thick blood film, malaria infection was diagnosed with a total of 2288 parasites/μL (2080 schizonts/μL). Mild anaemia was detected with haemoglobin of 10.8 g/dL. The patient was diagnosed with P. falciparum malaria based on a thick blood film with 336 parasites/μL (272 schizonts/μL). Mild anaemia was detected with haemoglobin of 9.5 g/dL.On February 27th 2020, a 39-year-old female presented to the OPD with history of fever and joint pain for 2 days and headache, weakness and low back pain for 4 days. Her axillary temperature on admission was 37.0 °C and she was diagnosed with uncomplicated P. falciparum/P. malariae mixed infection based on presence of 32 ring-stage parasites/μL and 520 schizonts/μL for P. falciparum and one trophozoite “band form” for P. malariae. Haemoglobin was 7.8 g/dL. The patient was diagnosed with uncomplicated mixed species malaria infection.On March 3rd 2020, a 2-year-old boy presented to the outpatient department with persistent vomiting, anorexia and headache. He had an axillary temperature of 38.3 °C. Microscopy slides were interpreted as P. falciparum malaria.On April 29th 2020, a 14-year-old male with mild symptoms of fever, headache, joint pain and anorexia presented to Nagongera Health Center IV, Tororo district. He had an axillary temperature of 37.4 °C. Malaria was diagnosed by microscopy with 4016 parasites/μL (96 schizonts/μL). Haemoglobin was 11.1 g/dL. The patient was diagnosed with uncomplicated P. falciparum infection according to national Ugandan treatment guidelines with artemether lumefantrine 20 mg/120 mg twice per day for 3 days and discharged home [P. malariae parasites after artemether-lumefantrine [P. malariae [®DNA Micro kit, cat numb. 56304) [Plasmodium speciation was performed using modified methods described by Snounou et al. [Plasmodium malariae parasite DNA was detected in material from all slides; P. falciparum was also detected for case 4 (Table All five cases were treated for uncomplicated ged home . Althougfantrine , 17, sugmalariae , 19. Thiu et al. . PlasmodP. malariae was the most likely cause of the clinical symptoms. All cases were classified as uncomplicated malaria, although they presented with anaemia, a condition that is often associated with chronic infection with P. malariae [P. falciparum infection was initially diagnosed. Only after an expert microscopist reviewed the blood slides was P. malariae identified as the infecting species. The presence of symptomatic P. malariae mono-infections has implications for the use of HRP-2 RDTs. In both of the study settings, HRP-2 RDTs are routinely used to diagnose malaria. Since non-falciparum Plasmodium species do not express HRP-2 antigen, at least four of these cases would presumably have been missed in routine practice and would not have received malaria treatment.In these five symptomatic malaria cases, malariae , 4. In fP. malariae cases reported here were older than typical P. falciparum cases in the area [P. malariae infections predominantly in children [Plasmodium infections using molecular diagnostic techniques is needed to quantify the burden of malaria in areas where multiple species may be present.he cases presented here all reported to the health facilities within a relatively short 13-week period. No effort is made to systematically investigate for non-falciparum malaria among individuals presenting with suspected malaria under standard care. This suggests that a considerable number of patients may present with uncomplicated non-falciparum malaria in this setting. The the area , which cchildren , 24, fur


The need to start treatment early for pregnant women who present with clinical features of malaria usually conflicts with the need to confirm diagnosis by microscopy (MP) before treatment, due to delays in obtaining results. Parasite sequestration in the placenta is also a problem. Rapid diagnostic tests (RDT), which detect soluble antigens, are a valuable alternative. The objective of this study was to evaluate pretreatment parasite prevalence by microscopy and by RDT and to assess the accuracy of RDT with MP as reference.A prospective cross-sectional study was carried out at the obstetrical unit of the Central Hospital in Yaoundé, during the period January-August 2015. Consenting patients with symptoms of suspected malaria in pregnancy were recruited and a blood sample taken for MP and RDT before treatment was started. The estimates of diagnostic performance  were calculated in OpenEpi online software using the Wilson’s score. The agreement, as reflected by the Cohen’s kappa, was calculated and interpreted using known intervals.Plasmodium falciparum. A Cohen’s kappa of 0.45 (95%CI: 0.26–0.64) was obtained, consistent with a moderate agreement between the tests.The results showed that, out of the 104 patients recruited, 69.2% (95%CI: 59.1–77.5) were MP positive while 77.94% (95%CI: 63.1–80.9) were RDT positive. The sensitivity of the malaria RDT was 91.67% (95%CI: 83.69–96.77) while the specificity was 53.13% (95%CI: 31.39–65.57). The diagnostic accuracy of the RDT with MP as reference was 79.81% (95%CI: 70.0–86.1). All cases were due to The diagnostic accuracy of the CareStart™ malaria Pf/PAN compared to microscopy was high, but not as desirable, with a false negative RDT at very high parasitaemia. In tertiary facilities, RDTs appear to provide a better diagnostic solution compared to microscopy. However, future studies with larger sample sizes should make this observation more generalizable; as missing a case could have serious consequences on pregnancy outcome.The online version contains supplementary material available at 10.1186/s12936-022-04109-6. Plasmodium falciparum  while a little more than one tenth had signs suggestive of other infections [13.5% (95% CI: 07.4–21.2)]. There were eight less positive cases by microscopy [69.2% (95%CI: 59.1–77.5)] than by the malaria RDT [77.9% (95%CI: 63.1–80.9)]. The geometric mean parasite load was 25,282 /µL among MP positive cases. The only .3–87.3. The indicators of diagnostic performance of the malaria RDT were calculated and summarized in Table P. falciparum (TPf)/uL, with a range of 457–10,000/uL. The same number, eight, was positive for RDT. Discrepant results were found in four cases. Two (14.3% of the 14 women) had negative RDThe sample T and positive MP (mean parasite load 457/uL) and 2 with negative MP had a positive RDT. Overall, seventeen women were negative for both MP and RDT, and only four of them (23.5%) had no symptoms or signs suggestive of other infection.Among the 14 pregnant women with signs suggestive of other infections, eight were positive for malaria by light microscopy and their mean parasite load was 1,896 trophozoites of The goal of this study was to determine how well a malaria RDT test performs in the diagnosis of malaria in febrile pregnant women in a tertiary health care facility in Cameroon. The study sample, composed of 104 pregnant women consulting at the maternity of the Central Hospital in Yaoundé, showed diversity in terms of maternal age, parity and gestational age, suggesting good representation. The high level of education with 90% of patients having attained secondary level of education or higher also reflects the general population in the urban setting of Yaoundé.The importance of IPT and LLITN in the prevention of malaria in pregnancy has been reported . The decAll our patients reported fever and it was confirmed in 79.8% of them, illustrating the value of fever as a key clinical feature of malaria in pregnancy in malaria endemic settings. More than half (61.5%) of our patients showed clinical signs of severity including hyperpyrexia (temperature of 40 °C and above). Although this does not figure on the WHO criteria for severity of malaria of 2010, it may be considered crucial in the context of pregnancy, where high temperatures could lead to fetal death.The accuracy of RDT with respect to MP (79.81%) is only slightly less than the 82.9% reported . The senThe geometric mean parasite load (2.73/uL) among the patients with negative RDT is high but much less than the value among those with a positive RDT . Patients with signs compatible with other infections had a sharp decrease in positive test results for RDT and MP (8/14 or 57% for both) and the average parasite load among those with positive MP dropped to 3,971/uL. These suggest a false clinical diagnosis for malaria in some of these cases. The low MP positive rate (1.9%) and low mean parasite load (457/uL) among patients with negative RDT and presence of signs suggestive of other infection favors this combination of arguments to rule out malaria in most of these cases. Submicroscopic malaria infections appear to be common in pregnancy, especially among the primigravidae as seen in recent studies , 35. IndP. falciparum was observed by microscopy and none of the PF + Pan RDT was positive for 'Pan ‘ and negative for 'Pf ‘, implying that none of these tests was positive for monoinfection with species other than P. falciparum. This is not consistent with earlier findings in the same setting and earlier reports of non-falciparum placental malaria, especially due to P. malariae [The geometric mean parasite load among MP positive subjects was high . However, this is about half the parasite load considered hyperparasitaemia for a malaria hyperendemic area . Only P.malariae , 41, andhrp2/3 genes, as has been shown in previous studies [One of the five patients with negative RDT and positive MP, had a parasite load that was too high to be counted. This ‘prozone effect’ has been reported in earlier studies . It may  studies  and needThese results should, however, be interpreted with some caution. Firstly, the small sample size of this study. It would have been desirable to have larger sample size to improve on the study power, although in prospective designs with large sample size requirement, cost may be a limiting factor. However, this study took place in the maternity of a tertiary facility where not as many malaria suspected pregnant patients are seen compared to district or primary health centers. Secondly, the number of symptomatic volunteers suspected for malaria among pregnant women is a smaller proportion of all symptomatic patients suspected for malaria in most health facilities in Cameroon. Thirdly, the specificity of the RDT test could be different (better) if the sample size was larger and design allowed for more sampling of pregnant women in the hospital outpatient and at antenatal care. Therefore, in the context of this study, the accuracy would seem to be biased towards positive cases. In addition, a significant number of pregnant women was excluded, some of whom should have been part of the study were they tested. They included symptomatic pregnant women who self-medicated before visiting at the hospital; or who were diagnosed for other febrile conditions than malaria, further limited the number of negative cases. Therefore, this study sample reflected a typical scenario under the local malaria management conditions. Future studies will consider these factors carefully when estimating an appropriate sample size for diagnostic evaluations in this special patient population.This study has shown slightly higher positivity for malaria RDT than for microscopy in pregnant women presenting with fever in a tertiary care setting in Cameroon. This may not, however, necessarily mean greater sensitivity, as patients with very high parasite densities could potentially present negative RDT results. Owing to the advantages of promptitude of results and little need for laboratory, RDT is recommended for facility diagnosis of malaria among pregnant women in the maternity. Studies with larger sample sizes are needed to make the findings more generalizable; as missing a case in pregnancy could have serious consequences on maternal or child health.Additional file 1: Table S1. Parasite load with respect to TDR and MP results. Table S2. MP and TDR among patients with signs compatible with other infections.


Plasmodium vivax malaria is one of the most lethal infectious diseases, with 7 million infections annually. One of the roadblocks to global malaria elimination is the lack of highly sensitive, specific, and accurate diagnostic tools. The absence of diagnostic tools in particular has led to poor differentiation among parasite species, poor prognosis, and delayed treatment. The improvement necessary in diagnostic tools can be broadly grouped into two categories: technologies-driven and omics-driven progress over time. This article discusses the recent advancement in omics-based malaria for identifying the next generation biomarkers for a highly sensitive and specific assay with a rapid and antecedent prognosis of the disease. We summarize the state-of-the-art diagnostic technologies, the key challenges, opportunities, and emerging prospects of multi-omics-based sensors. Malaria is a significant public health concern that continues to claim the lives of more than 435,000 people each year. The antimalarial drug resistance and detection of low parasitemia form a primary barrier to achieving the United Nations Sustainable Development Goals of ending malaria epidemics by 2030 . This aiP. falciparum specific}, pLDH {P. vivax, P. ovale, and P. malariae-pan} [The advancement in diagnostics can be grouped into two categories: technologies-driven and omics-driven . The adviae-pan} , aldolasiae-pan} ) in the iae-pan} , and theiae-pan}  for diffiae-pan} ,14,15,16iae-pan} ,18,19 aniae-pan} ,21 have iae-pan} ,24,25,26iae-pan} ,28,29, bIn short, we will discuss the recent development of omics-based malaria biomarkers ,32,33,34Plasmodium pathogenic species can be performed in clinical settings using phenome assays such as light microscopy-based diagnosis and differentiation of Plasmodium species [P. falciparum as compared to in P. vivax) [P. falciparum-iRBCs sticky leading to severe clinical complications such as anemia and cerebral malaria [Diagnosis of  species . The inf species ,50, flow species ,52, magn species , or dete species  gradient. vivax) , loss of malaria , and enh malaria  creating malaria . These d malaria  or enric malaria ,58,59 fo malaria ,61.Plasmodium species with high accuracy and sensitivity by nested-PCR targeting of ssrRNA [Chew et al., Malpartida-Cardenas et al., and Hede et al. have reported a nucleic acid-based mode of diagnosis of f ssrRNA ,63,64. Nf ssrRNA ,67,68,69f ssrRNA ,73,74,75f ssrRNA ,77,78 off ssrRNA . In manyf ssrRNA .P. vivax and P. falciparum, respectively [P. falciparum [P. vivax due to the lack of continuous in vitro culturing property. Swearingen and Lindner, 2018, Swearingen et al., 2017, and Lindner et al., 2019 have reported a few studies to attempt proteogenomics of P. vivax and P. falciparum to compare the salivary stage of the respective parasites [Proteomics of parasite pellet and parasite secreted proteins in plasma give abundant parasite proteins used for diagnostic application ,20,21. Aectively . Metabolectively  or erythlciparum . The toolciparum . This inarasites ,87,88. Aarasites . The omiP. falciparum [P. vivax, one cannot categorize the malaria infection as severe or non-severe [However, the omics-based approaches have their limitations  and henclciparum , that hin-severe .P. vivax and P. falciparum belong to the same genus with considerable differences in their G + C content in genetic material and their capability to infect hepatic cells to form hypnozoites, adaptability towards antimalarial drugs, and ability to grow in vitro continuously.Both parasites, Plasmodium vivax (Pv) exhibits low parasitemia in the host and has an uncharacterized genome and proteome due to a lack of knowledge of how to culture P. vivax in vitro. For several decades, the hepatocytic stage of P. vivax has failed to grow in in vitro conditions. Sangiamsuntorn K et al. [P. vivax culture remains a roadblock in understanding its pathobiology [P. vivax undergoes the quiescence known as hypnozoites in the host liver, which is responsible for disease relapse. The major impediment is that we lack a method or instrument capable of detecting hypnozoites to preclude the relapse. Primaquine and Tafenoquine  are the most effective drugs against hypnozoites of P. vivax. However, it leads to severe-hemolytic conditions leading to death in classes 1 and 2 G-6-PD deficient patients. This makes the detection of G6PD deficiency in the patient an essential prerequisite in vivax malaria treatment.K et al.  and YongK et al.  successfobiology ,93. AddiP. falciparum is very well studied and has a parasite protein for its diagnosis and differentiation. However, P. falciparum has started deleting HRP2/3 genes, differentiation biomarkers, from the genome, suggesting that the role of the gene might not be essential for the pathogen’s survival [On the other hand, survival . It has Recent advancements in diagnosis techniques have also enabled the in-depth exploration of the biomolecules including a higher resolution and increased accuracy and sensitivity . The othBox 1P. vivax and P. ovale.Malaria parasites show a vast difference on their molecular level resulting in unique life cycle stages such as hypnozoites in There is a paucity of accurate, reliable, rapid, and pathogen species differentiating diagnostic assays in endemic regions of the world.Delayed diagnosis contributes to the augmentation of resistant strains and antimalarial drugs facilitate the selection of mutants, aggravating the condition.Advancements in malaria diagnosis are propelled by advancements in technology and omics fields.The dearth of understanding about the pathogen results in a scarcity of promising diagnostic or therapeutic targets.Omics-based understanding of the pathogen or host response may facilitate understanding of the pathogenesis.Integration of multiple omics facilitates a holistic view of pathobiology and highlights crucial biomolecules for diagnostic and therapeutic purposes.Phenome-based diagnostics have advanced by using whole blood cells on a microarray chip to stain iRBCs using nuclear staining dye and correlating it with microscopy findings. The method is fast, easy to use, and has shown reliable outcomes .P. falciparum [P. falciparum along with Artemisinin resistance status in the infected pathogen strain [The nucleic acid-based diagnosis uses small subunit ribosomal RNA (ssrRNA) to amplify the genus and species-specific stretch to identify Plasmodium species . Loop-melciparum ,64. LAMPn strain . Hede etn strain . CRISPR n strain , and HPVn strain , and is n strain  diagnosin strain , differen strain  in malarP. falciparum malaria from P. vivax malaria [Proteomics-based assays, such as serum proteomics, have shown steady growth in the field of diagnostic applications for the detection of cancer and other human diseases with high sensitivity . It can  malaria . Protein malaria ,104,105. malaria ,21.P. falciparum-infected patients compared to healthy volunteers can also be used for a diagnostic marker search [P. vivax infected host [P. falciparum, whereas there is a significant alteration of retinol metabolism in P. vivax. Metabolites have also been reported to show the prediction of the antimalarial mechanism of action [Metabolome profiling of serum/plasma ,81,106 or search . A P. vited host  exhibitsf action  and provf action ,109,110 f action  have repf action .P. falciparum and P. vivax, P. falciparum is well studied, resulting in integrated omics, transcriptomics, and proteomics analysis to understand different stages in infected hosts [P. falciparum extracted from the salivary gland of mosquitos exhibited the role of a set of genes that help in parasite maturation and are capable of infecting the liver in humans [P. vivax due to limited information. Swearingen et al. reported using a proteogenomic tool to prepare a customized database of P. vivax isolated from the Thai population. The study explains the similarity and differences between P. vivax and P. falciparum proteomes [The integrated omics approach helps unravel the intricacies of pathobiology, given that the required databases and tools are available to the researcher. Of ed hosts . Proteoged hosts . Transcrn humans . Howeverroteomes . The metroteomes . HoweverPlasmodium parasite, which can be confirmed by using microscopy [The infected human exhibits symptoms on exposure to a disease that results from the alteration of the biochemical, physiological, or combination of both. The understanding of the disease is first looked at based on physiological alterations such as RBCs morphology change in the presence of the croscopy . The altcroscopy . Understcroscopy . This incroscopy ,114 and croscopy . KnowingP. vivax; however, each omics individually has its limitations. For example, the phenome of the infected cells is altered in terms of formability, rigidity, and adhesive character. However, these phenotypes can be due to other factors and micro-environmental variations such as sickle cell anemia [Multiple omics enhance perceptiveness towards an unknown organism such as l anemia , other il anemia  leading l anemia . Diagnosl anemia .On the other hand, genomic research has led us from predicting genotype by studying phenotype  to targeIn contrast, metabolomics is easy to extract but is highly dynamic, unlike proteins/antibodies, which have a higher self-life resulting in false positives even after the infection is clear from the system. However, handling metabolites is a challenging task; due to their highly dynamic nature, they tend to convert to their byproducts if not ceased properly. There are tools such as Qemistree that look promising in retracing the molecular signature back to predict the precursor metabolite , but it Mycobacterium tuberculosis diagnosis [Diagnostic and prognostic biomarkers are required with higher accuracy, longer shelf-life, and faster turnover rate. It is necessary to have a panel of biomolecules from host and parasite to accurately detect the causative agent and its antimalarial profile, such as geneXpert in iagnosis . The posiagnosis  and CPTAiagnosis ,127,128 The Foundation for Innovative New Diagnostics (FIND), along with WHO as a collaborating centre, has focused on laboratory strengthening and diagnostic technology evaluation since 2003. Utilization of funding to translate the findings to the clinics where the diagnosis, differentiation of parasite species, prognosis, and antimalarial profile can be screened in the minimum time with the highest accuracy and sensitivity is crucial to provide a better quality of life in a malaria-prone population. The dormant stage of vivax diagnosis coupled with a quantitative assay for G6PD testing or polymorph confirmation is necessary to explore the distribution of classes of G6PD deficiency in the population followed by Primaquine treatment. Identifying dormant vivax carriers and the asymptomatic patients may lead to a targeted approach to eliminate the vivax reservoirs. Proper classification of non-severe and severe vivax malaria is crucial in a timely prognosis for effective treatment . These aBox 2A biomarker or tool is necessary for the differentiation of malaria-causing pathogen species in humans with high sensitivity and specificity.A biomarker or methodology is necessary for the diagnosis of asymptomatic malaria patients and hypnozoite carriers for eliminating the parasite reservoir from the host.P. vivax parasite for comprehensive analysis of the pathogen due to low parasitemia in clinical samples.There is inaccessibility of the P. vivax continuously in the in vitro cell culture condition.There is an inability to grow P. vivax is one of the major roadblocks in the quest for a diagnostic, differentiation, and prognostic biomarker search.The paucity of complete proteomic and metabolomic databases of P. vivax. Most of the genes are hypothetical, uncharacterized, or unannotated.There is a lack of annotation of the genome of There is a lack of clinical information with heterogeneity information such as gender, race, age, and geographic location to understand the vivax malaria severity.There is a lack of information on the correlation between parasitemia and the severity of vivax malaria.P. vivax for the efficient treatment and elimination of the parasite reservoir from the host system.The coupled diagnosis of G-6-PD deficiency class along with P. vivax information is the source of extrapolation for the understanding of P. falciparum. Hence, the need has peaked for a short-culture-based multi-omic for the patient. Multi-omic information may help diagnostics and therapeutic modalities on a mass screening of vivax malaria from mixed or other parasite species in endemic regions. Additionally, the biosensor development towards the ultra-low parasitemia detection to check the correlation of parasitemia and severity for prognostics purposes, along with a global repository for the curated data of the clinical conditions, might help in understanding the heterogeneity involved in providing global/customized statistically significant biomarker candidates using artificial intelligence. This will help achieve the goal of timely diagnosis and efficient treatment towards the UN mission of global malaria elimination.The advancements in the field of diagnosis concerning the development of technology on the omics-driven front have facilitated highly sensitive and specific diagnostic prototypes ,114, but


We use nonlinear mixed effects modeling to establish malaria protective PPQ levels and risk factors for suboptimal protection. Compared to DP every 12 weeks, DP every 4 weeks is associated with 95% protective efficacy (95% CI: 84–99%). A PPQ level of 15.4 ng/mL reduces the malaria hazard by 95%. Malnutrition reduces PPQ exposure. In simulations, we show that DP every 4 weeks is optimal across a range of transmission intensities, and age-based dosing improves malaria protection in young or malnourished children.Intermittent preventive treatment (IPT) with dihydroartemisinin-piperaquine (DP) is highly protective against malaria in children, but is not standard in malaria-endemic countries. Optimal DP dosing regimens will maximize efficacy and reduce toxicity and resistance selection. We analyze piperaquine (PPQ) concentrations ( Intermittent preventive treatment with dihydroartemisinin-piperaquine (DP) is protective in children against malaria. Here, the authors analyze plasma drug concentration, malaria incidence, and drug resistance markers from a clinical trial in Uganda and determine the optimal DP dosing regimen. Plasmodium falciparum, including severe malaria and death1. In Uganda, nearly 75% of infants in one study developed malaria before 1 year of age2, and by 2 years of age, an average malaria incidence exceeding 6 episodes per year has been reported3. Prompt effective malaria treatment, long-lasting insecticidal bednets (LLINs), and indoor residual spraying of insecticides (IRS) have been the mainstays of malaria control for young children, accompanied by decreases in the global malaria burden1. However, reductions in malaria incidence and mortality have stalled, and new malaria control interventions are needed1.In malaria-endemic regions young children bear the greatest burden of malaria, primarily due to 5. For children residing in most of Africa, where SP resistance is already widespread and malaria transmission is perennial, a highly effective WHO or country-approved IPT regimen is not available, and in Uganda IPT with SP was ineffective6. A novel IPT drug regimen is needed for African children.Intermittent preventive treatment (IPT), in which full antimalarial treatment courses are given at fixed intervals to prevent malaria, is utilized to reduce malaria incidence in vulnerable populations. Seasonal malaria chemoprevention, in which children receive monthly sulfadoxine-pyrimethamine (SP) and amodiaquine during malaria transmission months, has reduced malaria incidence and mortality in children in parts of the west Africa7. Clinical trials among children in Uganda showed that monthly IPT with DP was superior to placebo or to DP every 12 weeks in young children, with protective efficacies >95%8. When administered to school-age children, IPT with DP every 4 weeks lowered community level parasite prevalence9. Large studies have not identified significant toxicities associated with IPT with DP in children, even though plasma PPQ concentration has been positively associated with lengthening of the corrected QT interval (QTc)11. Despite the appeal of DP for IPT, the optimal dose and frequency, particularly in children less than 2 years of age, is not well established. Underdosing of DP for IPT could result in inadequate preventive efficacy and selection of antimalarial drug resistance, while overdosing could increase cost and the risk of toxicity.The artemisinin-based combination therapy dihydroartemisinin-piperaquine (DP) is the leading candidate for IPT in young children in malaria-endemic regions with widespread SP resistance. Dihydroartemisinin rapidly clears parasites, while piperaquine (PPQ), the longer-acting aminoquinoline partner drug, eliminates residual parasites and provides sustained protection against new infections for approximately one month after treatmentP. falciparum markers of decreased antimalarial drug sensitivity.In this work, to gain insights into optimal DP regimens for IPT in young Ugandan children, we leveraged data from a randomized controlled trial and used pharmacokinetic/pharmacodynamic (PK/PD) modeling to describe the pharmacokinetics (PK) of PPQ and characterize relationships between PPQ exposure and risks of malaria, toxicity, and selection for markers of aminoquinoline resistance. Furthermore, we conducted simulations to quantify how optimized DP dosing regimens between 2 and 24 months of age would decrease malaria incidence, risk of QTc prolongation, and selection for Among the 280 children who received at least 1 course of DP, 243 (87%) completed follow up through 36 months of age : 0.05 [95% CI: 0.012–0.16]). The cumulative risk of malaria after receipt of DP was 8% (6.7–9.3%) through 84 days after dosing for DP every 12 weeks and 0.1% (0.0–0.30%) through 28 days after dosing for DP every 4 weeks. In the DP every 12 weeks arm, cumulative risk of malaria varied by transmission period, with high transmission periods in 2016 and 2017 associated with significantly higher risks of malaria compared to low transmission periods  with 2 transit absorption compartments provided the best description of the PK data  to represent liver enzyme maturation during early childhood, significantly modified PPQ clearance as shown in Eq. θCL is the population clearance, θEC50 is the PMA when maturation reaches 50%, and η is the between-subject random term , height for age z-score (HAZ), or weight for height z-score (WHZ), all reduced PPQ bioavailability . Malnutrition, as measured by WAZ provided the greatest statistical significance, the greatest covariate effect size (for each SD decrease in z-score there was a decreased bioavailability by 11.3% for WAZ vs 4.7% for HAZ or 4.4% for WHZ), and the best model fit by visual predictive check. Therefore, WAZ was selected as the covariate to represent malnutrition in the final PK model. In the final model, each standard deviation decrease in WAZ, decreased the relative oral bioavailability by 11.3%. In addition, when DP was self-administered there was lower PPQ oral bioavailability (ΔOFV −94.8). All three daily doses of DP were directly observed for participants in the intensive PK substudy, otherwise, the first DP dose was administered in the clinic, and the remaining 2 were given to the guardian to be administered at home. Compared to the intensive DP dosing episodes, oral bioavailability was 60% lower if the DP doses were taken at home . Transmission period and time-varying PPQ concentration were included in the final model as shown in Eq. 13, which would enable us to predict PPQ concentrations at the time of liver emergence. We found that in our study population, the semi-mechanistic model did not predict the data well, and the empirical model was used as the final model. Sex, IPT arm, maternal IPT regimen, WAZ, WHZ, and HAZ were not associated with the hazard of incident malaria.Three increases in malaria incidence corresponding to the annual high transmission periods in Tororo occurred Fig. , and we To assess relationships between PPQ concentration and risk of QT interval by Bazett’s correction (QTcB) prolongation, a PK-QTc model was developed based on data from the intensive PK substudy with paired ECGs from 32 participants at 32 and 104 weeks of age. As previously reported, the median QTcB pre-drug was 413 msec (range: 347–472), and post-drug was 424 msec (range: 388–482). Increasing PPQ concentration increased the QTcB as described in the following linear equation: QTcB = modeled baseline QTcB + [PQ] × 0.046/1000. Each 100 ng/mL increase in PPQ concentration was associated with a 4.6 msec increase in the QTcB , and in pfcrt, the gene that encodes the chloroquine resistance transporter (PF3D7_0709000). The following polymorphisms were evaluated: pfmdr1 N86Y, pfmdr1 Y184F, pfmdr1 D1246Y and pfcrt K76T14. Genotype data were available from 142 episodes of parasitemia (88% of eligible episodes) from 8 to 112 weeks of age ns Table  and predns Table . The agens Table . Finallyns Table . As peakWe report a PK/PD analysis based on the largest longitudinal clinical trial of IPT with DP in young children, including longitudinal pharmacokinetic, incident malaria, electrocardiographic, and drug sensitivity data. For children <2 years of age, we identified a malaria preventive PPQ concentration of 15.4 ng/mL. Children 1–2 years of age had the highest risk of malaria in the cohort, and had lower PPQ concentrations compared to children <1 year of age. Malnourished children were at particular risk of low PPQ exposure, with children with WAZ ≤ −2 predicted to have an increased incidence of malaria compared to better nourished children. Adherence to the three-day DP regimen was lower than expected, but despite low adherence DP given every 4-weeks had a protective efficacy of 95% compared to DP every 12-weeks. Based on these data, we predicted that a 4-week dosing interval with age-based, instead of weight-based, dosing would provide maximal protection and reduce disparities in PPQ exposure due to age and nutritional status.13 and Thai adults (20 ng/mL)12. Among highly malaria-exposed Ugandan pregnant women, a level of 10.3 ng/mL was associated with the prevention of both symptomatic and asymptomatic parasitemia15. Despite differences in age, immune status, and region, with potential differences in drug resistance patterns or population genetics, these PPQ targets are remarkably similar. We found that median PPQ trough concentrations in young children were at or below 10–20 ng/mL with DP dosed every 4 weeks. As a result, we would expect that even a short delay in DP dosing frequency beyond 4-weeks would result in a loss in protective efficacy. Similar challenges have been observed with SMC implementation with SP with amodiaquine, where malaria incidence reached levels similar to no chemoprevention by 29–35 days after a round of SMC 16.PPQ concentrations predict malaria protective efficacy in a variety of high-risk populations receiving chemoprevention with DP, and identifying protective concentrations is essential to our effort to expand the role of DP to IPT. The 15.4 ng/mL protective PPQ target derived from this study is similar to protective levels against symptomatic malaria identified in children <5 years of age receiving DP for seasonal malaria chemoprevention in Burkina Faso (12.9–17.5 ng/mL)18. A variety of biomarkers of acute malnutrition including mid-upper arm circumference (not available for this study), WHZ, and WAZ have been linked to lower antimalarial drug exposure for malaria treatment. More research is needed to elucidate the pathophysiologic mechanism for these findings20. Several physiologic changes due to malnutrition have been implicated, including increased intestinal inflammation leading to reduced drug absorption and low plasma albumin concentrations resulting in altered protein binding21. We found that even small reductions in WAZ lead to lower PPQ bioavailability. Although weight-based dosing strategies can incorporate dose increases to compensate for liver maturation in infancy, low weight malnourished children can fall into weight-bands designed for younger children, leading to unintentional underdosing22. This can be further exacerbated when children, as is the case of this study, between 1 and 2 years of age, were already underdosed.Malnourished children were at the highest risk of suboptimal PPQ exposure and were predicted to suffer the highest incidence of malaria when receiving IPT with DP. Lower PPQ exposure was related to lower oral bioavailability with malnutrition. Lower oral bioavailability has been linked to malnutrition, as defined by low WAZ, among children <5 years of age for lumefantrine and SPWe propose an age-based dosing algorithm that would both increase PPQ exposure in older children, compared to the dosing regimen used in the parent trial, and reduce the impact of malnutrition on PPQ exposure. Notably, we found that easily implementable revised weight or age-based DP dosing strategies did not fully eliminate the impact of malnutrition on PPQ exposure Table . Thus, c25. The parent clinical study was no exception. Although >95% adherence was self-reported among study participants, on average PPQ absorption was 60% lower when only 1 dose, compared to all 3 doses of DP was directly observed. Although PPQ absorption is heterogeneous between individuals and dosing occasions13 and can be increased modestly by food27, the magnitude of the association with self-administration is most consistent with a low adherence effect.By identifying malaria protective PPQ concentrations for young children, we predicted that if malaria transmission had been higher, such as would be expected in similar regions not receiving IRS, the incidence of malaria on IPT would have been higher. In the case of high malaria transmission, in addition to age and nutritional status, adherence would drive DP protective efficacy. Adherence is a well-known barrier to effective IPT, and low adherence has been observed with multiday malaria treatment and IPT regimens4. Incident malaria was clustered during three short periods of high transmission, each timed late after a round of government-implemented IRS . We recommend future IPT studies incorporate supplemental measures for drug adherence . Fourth, we did not implement a mechanistic model to estimate PPQ concentrations at the time of parasite emergence from the liver, which occurs some days prior to symptom onset. We explored back-extrapolating the time of parasite emergence using parasite density at malaria diagnosis and historic parasite replication rates in adults13, but found this mechanistic approach prevented us from predicting the clinical data. As all participants received the same three-day treatment courses, PPQ concentrations that prevent clinical malaria would correlate with PPQ levels at liver emergence, and as a result, we only simulated three-day treatment courses for our optimized regimens. Finally, we could not include maternal SES as a covariate on the baseline malaria hazard in the final PK/PD due to model instability. Fortunately, though SES status reduced the intraindividual variability in our exploratory models, it did not modify the key PK/PD relationships or baseline malaria hazard estimates during chemoprevention. Future studies should consider an externally validated SES measure.There were several limitations to this study. First, a placebo IPT arm was not included in the clinical trial, and thus the baseline hazard of malaria was dependent on data from the DP every 12-week arm adjusted for PPQ exposure. This approach may have under-estimated the baseline hazard of malaria during the study. Second, exposure to dihydroartemisinin, the short-acting partner drug in DP, was not quantified. To incorporate the parasiticidal activity of dihydroartemisinin into our model, we assumed cumulative survival returned to 100% when DP was given, as dihydroartemisinin resistance had yet to be detected in southeastern Uganda at the time of the study42. However, despite a high burden of malaria in countries like Uganda, IPT in children is not yet recommended in east Africa, where SP resistance is widespread and seasonal approaches are not appropriate. The results of the parent clinical trial and this large PK/PD analysis assessing the drug exposure-response relationship for PPQ and malaria protection, risk of QTcB prolongation, and drug resistance markers confirms that DP every 4-weeks in children <2 years of age is effective and safe, and can be further optimized by utilizing age-based dosing bands. An age-based DP dosing strategy could have additional operational benefits for IPT, by eliminating the need to weigh infants receiving DP. We also found PPQ exposure was lower in malnourished and children 1–2 years of age, and that an age-based dosing strategy would particularly benefit these children. Although DP every 4-weeks is highly effective for IPT in Africa, we show that there are simple and easily implemented dose modifications that could improve protection.Large studies of seasonal malaria chemoprevention with SP plus amodiaquine in west Africa have been associated with dramatic reductions in malaria incidence and mortality in children <5 years of age8. Neonates, born to mothers enrolled in a separate trial of IPT during pregnancy in Tororo, Uganda43, were enrolled at birth from October, 2014 to May, 2015, and followed for 36 months8. Informed consent was provided by the parent or guardian for every participant. The study protocol was approved by the Makerere University School of Biomedical Sciences Research and Ethics Committee, the Ugandan National Council for Science and Technology, and the University of California, San Francisco Committee on Human Research. The clinical trial registration number is NCT02163447.A randomized controlled trial provided data and samples for the analysis8.Children were randomized prior to birth and received DP every 12 weeks or every 4 weeks from 8 to 104 weeks of age  was obtained prior to the first dose of DP at 32 and 104 weeks of age, and 2–3 h after the 3rd daily dose of DP for all 32 children in the intensive PK substudy.PPQ concentrations were quantified from plasma obtained from finger-prick at pre-specified timepoints 12, 16, 20, 36, 44, 60, 64, 68, 96, and 100 weeks of age and from venipuncture at 40 and 104 weeks of age. At each time point samples were obtained prior to the subsequent monthly course of DP or placebo. PPQ concentrations were additionally quantified when an episode of malaria or asymptomatic parasitemia was diagnosed between 8 and 112 weeks of age. A subset of 32 individuals was enrolled in an intensive PK substudy which occurred at 32 and 104 weeks of age. As previously reported45. A coefficient of variation <10% for quality control samples was used. Qualitative interpretation of chromatographs to further evaluate BLQ samples was conducted by two experienced mass spectrometrists. BLQ samples with no observed peak, based on review by the mass spectrometrists, or which were measured after a prior BLQ measurement without interval dosing of DP were excluded from the analysis. If a peak was observed in a BLQ sample, it was considered as BLQ.PPQ concentrations were determined from plasma using high-performance liquid chromatography and tandem mass spectrometry (HPLC-MS). Plasma for PPQ concentration quantification was separated from whole blood at the time of collection, and frozen immediately for storage at −80 °C. Plasma was shipped on dry ice to the UCSF Drug Research Unit in San Francisco, CA. PPQ quantification was conducted by protein precipitation using 25 µL of plasma, followed by liquid chromatography and tandem mass spectrometry. Calibrated standards ranging from 0.5 to 50 ng/mL were used and quality control (QC) samples comprise 1.5, 20, 40 ng/mL for sparse samples and standards from 10 to 1000 ng/mL and QC samples comprised of 30, 200, and 800 were used for the intensive PK analysisP. falciparum DNA by loop-mediated isothermal amplification (LAMP) as part of the parent study8. For the first new episode of parasitemia detected after a round of DP, genotyping for pfmdr1 N86Y, pfmdr1 Y184F, pfmdr1 D1246Y, and pfcrt K76T was conducted. Parasite DNA was extracted from a dried blood spot using Chelex-100, the gene of interest amplified using nested polymerase chain reaction, and polymorphisms detected using a ligase detection reaction-fluorescent microsphere assay46. A mutant infection at each locus was defined as detection of a mutant genotype, with or without concurrent detection of a wild-type genotype for a polyclonal infection. A wild-type infection was defined as detection of only pfmdr1 N86, pfmdr1 Y184, pfmdr1 D1246, or pfcrt K76 in the P. falciparum positive sample.A blood spot was collected and stored on filter paper at all routine visits and if malaria was diagnosed at an unscheduled visit. DNA was extracted from dried blood spots using Chelex-100, and all microscopy negative samples were tested for the presence of 47. Model building was guided by the likelihood ratio test to determine statistical significance, diagnostic plots, and internal model validation techniques, including visual predictive checks 48.All analyses were conducted in NONMEM version 7.4 or R version 3.6.1. We first established a model for venous plasma PPQ concentrations, followed by the addition of capillary PPQ concentrations to develop a joint model. We investigated 2-, 3-, and 4- compartment PK models linked to a first-order absorption model with lag time or absorption described by pre-specified transit compartments. Individual parameters were assumed to be normally distributed, and proportional and additive errors were evaluated for quantification of residual variability. Linear and log-linear models with and without an intercept were explored for the relationship between capillary and venous plasma PPQ concentrations. Clearance and volume parameters were allometrically scaled for bodyweight a priori by normalizing the child’s weight to the median weight of the study population (8.6 kg) and raising to the power of 0.75 for all clearance parameters and to the power of 1 for all volume PK parameters. Relationships between pharmacokinetic parameters and covariates  were assessed by graphical inspection and formal stepwise covariate model building. Validated methods for incorporating BLQ PPQ concentrations including the M1-7 methods were exploredCox proportional hazard models were used as an initial evaluation of the raw data for cumulative malaria hazard by treatment arm. A parametric survival model, adjusted for repeated events was developed as the final model to predict the primary outcome, incident malaria. An incident malaria episode was defined as fever and positive blood smear >14 days from a prior episode of malaria (to minimize the effect of artemether-lumefantrine treatment failure). Exponential, Weibull, and Gompertz distributions were tested as the survival baseline model prior to evaluating covariates. Covariate analysis included time-varying PPQ concentration as defined by model-derived individual PK parameters, high malaria transmission period , age, sex, time-varying WAZ, time-varying HAZ, time-varying WHZ, maternal IPT regimen during pregnancy, and maternal SES. Covariate relationships for continuous covariates included linear and nonlinear relationships . Model building was guided by the likelihood ratio test, diagnostic plots, and visual predictive checks.49; and a novel age-based dosing regimen where DP doses were increased at 6 and 18 months of age for all children, regardless of weight. An age-based regimen was chosen as prior studies had identified malnutrition as a risk factor for under-exposure for PPQ in some populations and SMC is dosed by age to assist with large-scale implementation efforts51. For each regimen and adherence pattern , the final PK model was simulated 1000 times and the final parametric survival model for malaria hazard was simulated 10,000 times using the full monthly demographic data from a combined dataset of the 280 study participants from 8 weeks to 24 months of age which data contributed to this study and from 576 children from 6 to 24 months of age enrolled in a previously conducted set of clinical trials in Tororo, Uganda6. The percentage of time above the model-derived protective PPQ concentration per DP treatment course was estimated for each regimen from the final PK model. Simulations of the final parametric survival model were conducted using baseline hazards from 0.5 to 8 episodes per person-year. Malaria hazard was kept constant in the simulations.The PPQ concentration associated with protection from malaria was defined as the median PPQ concentration predicted to provide a 95% reduction in hazard of malaria as compared to no drug. Monte Carlo simulations were conducted to predict malaria incidence and time above protective PPQ concentrations under varying malaria transmission intensities and under different DP dosing regimens based on weight-band and age. The simulated regimens are listed in Supplementary Table 44. The corrected QTc by Bazett’s formula  associated with decreased sensitivity to chloroquine and amodiaquine and (b) selected by chemoprevention with PPQ52. The genotypes of the first new episode of parasitemia detected after a round of DP were included in the analysis. The analysis was conducted using logistic regression, and linear and Emax relationships were explored for the relationship between PPQ concentration and the probability of detecting a mutant parasite.A PK-resistance genotype model was developed to quantify relationships between PPQ concentration at the time of Further information on research design is available in the Supplementary InformationPeer Review FileReporting Summary


The quality of malaria test results is crucial for optimal patient treatment and care. The Ghana Health Service is successfully shifting from presumptive clinical diagnosis and treatment of malaria to the Test, Treat and Track (T3) initiative. In line with the initiative, the National Malaria Control Programme (NMCP) set out to improve the capacity of medical laboratory professionals in Ghana through a five-day Malaria Diagnostic Refresher Training (MDRT) to build competencies and skills in malaria diagnosis, especially in the three components of microscopy: parasite detection, species identification and parasite quantification. This study evaluates the impact of the training on malaria microscopy.The training which was based on the World Health Organization basic malaria microscopy training guide employed presentations and practical approaches to malaria diagnosis. A total number of 765 medical laboratory professionals from various health facilities across the country were trained every other year from 2015 to 2019 and were included in this evaluation. Evaluation of this training was done using pre-test and post-test microscopy scores. The Negative Binomial fixed effect model was used in determining the overall effect of the training in improving the competencies of the participants on malaria microscopy.The ability of the medical laboratory professionals to correctly detect malaria parasites increased significantly from a median score of 64% prior to the training to 87% after the training (p < 0.001). The competencies of the medical laboratory scientists to correctly identify malaria parasite species and quantify the number of malaria parasites increased significantly from a median score of 17% and 20% pre-test to 78% and 50% post-test, respectively (p < 0.001). The results showed that participants’ competency level and skill to perform malaria microscopy  increased by approximately two folds after the training compared to the no-training scenario .The MDRT programme significantly improved participants’ performance of malaria microscopy over a short period of time. Though significant efforts have been made in an attempt to reduce burden, malaria continues to be a global health burden with an estimated 229 million cases and 409,000 deaths reported worldwide in 2019, with the hardest hit regions being the World Health Organization (WHO) African Region, which accounted for 94% of the global malaria cases and 94% of the global malaria deaths [Plasmodium lactate dehydrogenase (pLDH) test kits while microscopy costs up to 0.5 USD per slide [Plasmodium ovale can form latent intrahepatic hypnozoites that may cause disease several months or even years after the primary infection [As part of measures to reduce the malaria morbidity and mortality burden, Ghana rolled onto the test, treat and track T3) initiative of the WHO in 2010  initiati. The RDTer slide . On the er slide . Accurater slide . A falseer slide  whereas Plasmodium species with the exception of Plasmodium falciparum, inaccurate estimation of the parasite density; and the non-detection of mixed infections and low parasitaemia [Lack of training and limited supervision has led to poor characterization of the various sitaemia . While ssitaemia –14. One sitaemia . The Natsitaemia . In ordeThis study was a retrospective interventional study where the pre-test and post- test microscopy results of all the participants who took part in the malaria diagnostic refresher training for 2015, 2017 and 2019 were collected and analysed.The training was conducted in all the ten regions of Ghana. Each region is made up of districts with private and public health facilities such as Regional (secondary) and District (primary) hospitals; Health centres and Polyclinics as well as Mission hospitals, Quasi-government facilities and Private hospitals and laboratories. Participants were selected from these facilities to participate in the Malaria Diagnostic Refresher Training.The training consisted of a five-day residential intensive session, which was based on the WHO basic malaria microscopy learner’s guide . The traThe trainees were medical laboratory professionals belonging to the grade of either medical laboratory scientists (MLS), Technical Officers (TO) or medical laboratory assistants (MLA). They were selected by their respective Regional medical laboratory scientists based on their training needs. The number of participants per training session was a minimum of 20 to a maximum of 30 laboratory professionals per region.Facilitators on the other hand consisted of WHO certified malaria microscopists (Level 1 and 2), Nationally certified malaria microscopists (NCAMM level A and B), Regional malaria supervisors as well as National malaria supervisors from the National Malaria Control Programme and Clinical Laboratory Unit of the Ghana Health Service.The National competency assessment for malaria microscopy (NCAMM) programme is organized in accordance with WHO standards using the WHO Quality Assurance (QA) manual version 2  as a guiThe training was made up of theoretical presentation sessions and practical sessions as outlined in the timetable Table . The timFor the practical sessions on blood film preparation and staining, each participant was provided with 10 frosted-end microscope slides where they used one slide as a spreader to prepare 9 thick and thin blood films on same slide and selected the best 5 films for staining and assessment. The trainees volunteered for their blood samples to be collected as part of practical blood collection training while permission was sought from the head of laboratory of the nearest health facility to obtain blood infected with malaria parasite and it was granted. All other supplies for blood collection and quality malaria blood film preparation and staining were provided to participants as part of the supplies arrangements for the training.For the practical session on malaria RDTs, the participants were paired and provided with RDT kits and supplies for testing. The RDT tests were conducted by all participants on their respective pairs and also assessed as part of the simulation exercise. The malaria positive blood sample was also tested using the RDT to demonstrate positivity. Facilitators assessed the tests by marking the theory with the right answers, assessing prepared and stained blood films against the WHO criteria for assessing blood films as outlined in the WHO QA manual and scored. The thick film was measured with a ruler to assess if it was 12 mm in diameter and whether news print can be read under it before staining; after staining, it was observed to ascertain if 90% of the smear was intact. The distance between the frosted end and the thick film as well as that between the thick and thin film was examined to check if it was 10 mm; the thin film was assessed for a distinct head, body and tail. Microscopically, the thin film was assessed for the presence of a monolayer of red blood cells, while the thick film was examined for completely lysed red blood cells; finally, the staining was examined by assessing the colour of the white blood cells and red blood cells in the thin and thick film respectively. This was to establish if the staining clearly distinguishes the various stages of the parasite if present and the white blood cells from the background. The microscopy examinations in terms of parasite detection, species identification and parasite quantification were scored using a computerized excel database.P. falciparum, P. ovale, and Plasmodium malariae as well as low density (169–277 parasites per microlitre of blood), medium density (1789–2921parasites per microlitre of blood) and high-density . The slide bank is composed of over 6000 slides of different species which were internationally validated according to WHO protocols in developing a malaria slide bank as outlined in the WHO QA manual. Universal Power Saver (UPS) devices were also connected in place to avoid power disruptions during power outages before power generator plants at training venues were turned on.Each participant was provided with a light microscope, Immersion oil, two tally counters and a calculator for the examination of validated slides. The validated slides for practical microscopy parasite detection, species identification and parasite quantification were obtained from the National Archive for Malaria Slide Bank in Ghana composed mainly of the three species The morning session involved preparation of blood films, theoretical presentations and a review of the previous days’ microscopy assessment whiles the afternoon session entailed practical microscopy examinations (post-test assessments) staining of prepared blood films and another theoretical presentation. Each new day began with a presentation of the results and an open and intensive review of the slides examined the previous day where participants were freely allowed to share their opinions.P. falciparum, two P. malariae, two P. ovale, one mixed infection of P. falciparum and P. malariae and another one with P. falciparum and P. ovale, one low density, two medium density, one high density and five negative slides with time limit of 10 min per slide using the light microscope. This was to measure their capacity in parasite detection, species identification and parasite quantification.There was pre-test on practical preparation of blood films, followed by a theory pre-test consisting of questions on malaria parasitological diagnosis and pathogenesis. The participants were then taken through a presentation on use, care and storage of the microscope to introduce amateur trainees to the microscope to avoid any misuse and damage; after which they then took a practical malaria microscopy pre-test where they examined a set of 18 slides comprising three The blood films that were initially prepared were later stained. The objective of conducting these pre-tests was to assess the baseline level of each participants’ theoretical knowledge and practical skills in blood film preparation and staining; and in malaria microscopy.The facilitators used the criteria for assessing blood films as outlined in the WHO QA manual to assess the blood films, and graded them according to the prescription in the manual. The results of the slide examinations were scored at the end of each day’s activities and activities for the next day was then planned. This presentation paved way for the practical review of the pre-test validated slides. In this session, there was an open presentation of the performance of each trainee with respect to the previous days’ slide examination prior to the review of the slides. This was to allow the participants to share their opinions and recognize some of the mistakes they made during the slide examinations; it was also to identify gaps in the trainees’ microscopy skills and competencies. Afterwards, the slides were made available according to their compositions for participants to examine according to their needs and to fill the gaps identified with the guidance of the certified malaria microscopist.On the final day of the training the pre-test and post-test results for the theoretical assessments, blood films and microscopy examinations for each trainee was displayed and each trainee presented with a certificate of participation.The microscopy assessments were conducted under examination conditions where no talking or sharing was allowed and each participant was given 10 min to examine each slide for parasites and species identification or parasite quantification. Different sets of slides with similar composition were used for each of the tests. There were separate pretest slides for parasite detection, species identification and parasite quantification.A pre-test was conducted before the start of the training and a post-test after the training. Effectiveness was assessed by a difference in the performance of participants in these two tests.The trainees’ microscopy examinations were scored and graded using a standardized computer-based template; each score was presented over a hundred percent (100%). The emphasis of this study was on malaria parasite detection, species identification and parasite quantification among the laboratory professionals over a five-year period from 2015 to 2019.The trend differences in the outcome of the training in relation to some of the demographic characteristics of the participants such as cadre, previous in-service training experience, sex and facility type were assessed in a univariate analysis whiles the Negative Binomial and Poisson fixed effect models were used in finding the net effect of the training on the performance of the participants.The outcome of the trainee results was set premised the WHO grading system.Parasite detection: this is the ability of the laboratory professional to indicate whether a malaria parasite is truly present (sensitivity) or truly not seen (specificity). Each microscopist must obtain an 80% or more score in sensitivity and specificity to be deemed competent in parasite detection.Species identification: even though there are five types of Plasmodium species infecting humans worldwide, there are only three found in Ghana: P. falciparum, P. ovale and P. malariae. The participants are to know and differentiate between the four developmental stages of each Plasmodium species, namely the ring, trophozoite, schizont and gametocyte. The participants must also be able to differentiate between the parasites using their morphological features. A participant must score 80% or more in species identification.Parasite quantification: the detected malaria parasites must now be quantified. This is done in the thick film by counting the number of asexual stages of the malaria parasites per field, while counting white blood cells alongside until a total of a minimum of 200 WBCs are counted. In the thin film, parasite quantification is enumerated by counting the number of infected red blood cells alongside a total minimum of 5000 RBCs. Trainees must attain a score of 40% or more out of the total number of slides examined to be deemed quantifying accurately. Each of the internationally validated slide falls within a certain predetermined range; the trainees are expected to achieve a count of ± 25% of the count range of each slide as determined by the international validators.The pre and post-test microscopy results of all the participants who took part in the MDRT for 2015, 2017 and 2019 were retrieved and entered into a Microsoft Excel 2016 spread sheet and exported to Stata IC version 16 . Descriptive statistics  were used to quantify categorical variables. The median and interquartile ranges were used to summarize the pre- and post-test scores since the scores were heavily skewed . The Wilcoxon matched-pairs signed-rank test was used to compare the differences in median scores before and after the training. The Poisson and Negative Binomial fixed effect models with robust standard error were used in determining the net effect of the training on the knowledge score of the participants. The likelihood ratio test was used to test for overdispersion  in the count outcomes (discrete scores obtained by each participant). However, for the purposes of comparing effect size estimates, rate ratios from both models were reported. Sub-group analysis of the impact estimate was conducted to ascertain how the impact estimate varies across the years of implementation of the intervention, administrative regions, the different cadre of laboratory professionals as well as different health facilities. All statistical analyses were conducted using Stata 16  and p-values less than 0.05 were considered statistically significant.A total of 765 laboratory professionals from the 10 regions of the country comprising 613 males and 152 females of different cadres were enrolled in the study as shown in Table There was an overall improvement in the percentage median score for the participants after they had taken part in the training. Before the training was carried out, the median percentage score of the ability of the participants to correctly detect the presence or absence of a malaria parasite was 64%, however, the median percentage score increased to 87% after the training  in diagnosing malaria; some health centres may even not have microscopes in their laboratories. Participants from the private health facilities may also have recorded low pre-test and post-test performances because most of them are not usually exposed to new trends and guidelines in malaria microscopy; more so, most of them may not have the opportunities to partake in in-service trainings in malaria microscopy. A study in Ethiopia  reportedThe use of microscopy in diagnosing malaria through parasite detection, species identification and quantification are thought of as the gold standard. Accurate examination of a slide, however, requires highly skilled and competent medical laboratory professionals, especially when parasite densities are low. The analysis of results from this study, showed an improvement in the parasite detection skill of the participants with the difference in the median pre-test and post-test scores being 23%. However, a pre-test median score of 64% showed that most of the laboratory professionals had a degree of parasite detection skills before they took part in the training. Similar studies conducted in other African countries presented similar findings. While the reports of ten-day and seven- day trainings held in Nigeria presented mean pre-test detection scores of 53.9% and 48.9% with mean post-test detection scores of 70.7% and 56.8%, respectively , 21, an P. falciparum species is the commonest and most fatal malaria causing species in this country so the other species are seldom seen, therefore, making identification of the morphologic features of the other parasite species quite difficult. Other studies have also revealed that medical laboratory scientist have a difficulty in differentiating malaria species microscopically [Species identification was observed to have had the most impressive improvement margin of 61 percentage points per this assessment. The pre-test median score of 17% was very low compared to 18.9% reported by Aiyenigba, Ojo (18) and 27.9% reported by Olukosi, Agomo (21) whereas the post test score of 78% in this study was higher than the post-test scores of 38.3% and 39.2% reported respectively in the other studies. The opically , 25.Conducting parasite quantification is vital in monitoring disease severity and tracking the efficacy of the anti-malarial drugs especially in severe hospitalized malaria cases. In this study the quantification scores saw an improvement of 30 percentage points (20% pre-test rising to 50% post-test); comparable to the observations made for species identification, parasite quantification scores in the current study were quite low at the beginning of the Malaria Diagnostic Refresher Training for reports of other trainings too. While one study in Nigeria  reportedThe differences observed in the pre-test and post-test scores of the participants could perhaps be due to the fact that the first basic skill in malaria microscopy is detection of the malaria parasite hence the ability of most participants to accurately report the presence or absence of a parasite; however, species identification and parasite quantification using the count method was introduced later by the WHO . With moDespite the findings of this study and their importance in informing policies regarding training of laboratory professionals for management of other communicable diseases, there are limitations, which are worth noting. Firstly, the data used for this evaluation did not capture variables such as age of participants, the number of working years and number of years with experience in malaria microscopy which could affect the performance of the participants and confound the training outcomes. Secondly, information on the cadre for the 2015 training group was not available however all other needed data including the pre and post-test scores were available for usage in this analysis. Therefore, the cadre analysis carried out in this study used only 2017 and 2019 data. Finally, only the immediate outcome of the training is being assessed, the medium to long-term impact of the training at the facility level is not being assessed in this study.The Malaria Diagnostic Refresher Training has been shown to greatly improve malaria microscopy outcomes of the participants within the five-day period of intensive training with respect to parasite detection, species identification and parasite quantification skills. Therefore, periodic malaria microscopy in-service training and perhaps a review of the malaria diagnosis content for training of medical laboratory professionals to include pre-service training as well as regular on-site training and supervision by the already trained professionals may be necessary to assist in producing highly skilled and competent medical laboratory professionals who can accurately and reliably diagnose malaria microscopically.