P. falciparum Histidine-Rich Protein 2 (PfHRP2) have diagnostic and prognostic value in severe malaria. We investigate the potential use of plasma PfHRP2 and the sequestration index (the ratio of PfHRP2 to parasite density) as quantitative traits for case-only genetic association studies of severe malaria. Data from 2198 Kenyan children diagnosed with severe malaria, genotyped for 14 major candidate genes, show that polymorphisms in four major red cell genes that lead to hemoglobin S, O blood group, α-thalassemia, and the Dantu blood group, are associated with substantially lower admission plasma PfHRP2 concentrations, consistent with protective effects against extensive parasitized erythrocyte sequestration. In contrast the known protective ATP2B4 polymorphism is associated with higher plasma PfHRP2 concentrations, lower parasite densities and a higher sequestration index. We provide testable hypotheses for the mechanism of protection of ATP2B4.Severe falciparum malaria is a major cause of preventable child mortality in sub-Saharan Africa. Plasma concentrations of  PfHRP2, in a cohort of Kenyan children suffering from severe malaria. They suggest that plasma PfHRP2 and the ratio of the plasma PfHRP2 to the peripheral parasite density (sequestration index) as powerful quantitative phenotypic traits for severe malaria.Numerous candidate malaria protective gene polymorphisms have been proposed. Here, Uyoga et al. investigate associations between malaria-protective red blood cell polymorphisms and total parasite biomass estimated from plasma concentrations of  Plasmodium falciparum malaria has been the dominant cause of child mortality in tropical regions of Africa and Asia for much of the last 5000 years1. As a consequence, malaria has played a major part in shaping the human genome through the selection of mutations that confer a survival advantage1. An estimated one-quarter of the variability in the overall risk of malaria is heritable, a figure that reaches one-third for severe and complicated infections2.3. Although many of these candidates have not held up to detailed scrutiny4 there is now strong evidence in support of some. The most convincing relate to red blood cells5 and are particularly plausible given the central role of the erythrocyte in the biology of malaria6.Numerous candidate malaria protective polymorphisms have been proposed during the 70 years since, through his “malaria hypothesis”, JBS Haldane first suggested that the high prevalence of β-thalassemia in the Mediterranean basin might be reflective of natural selection by malariaP. falciparum malaria in Kilifi, Kenya. We found significant evidence in support of polymorphisms in 15 genes: seven  involved in red cell structure or function, seven  non-red blood cell protein coding genes, two of which related to immune pathways, and one non-coding RNA gene (LOC727982)7. Although all were associated with a lower risk of severe malaria, only three were associated with lower peripheral parasitemia. Parasite densities were approximately fivefold lower in both heterozygotes and homozygotes for the rs334 A>T allele in HBB, the βs mutation which encodes for the structural hemoglobin (Hb) variant HbS, in comparison to wild-type normal children, while they were marginally lower in both children with blood group O and in homozygotes for the derived allele at rs1541255 A>G in ATP2B4.Through a recently conducted case-control study, we investigated associations between mutations in a wide range of candidate genes and the risk of severe P. falciparum infections because of the time-varying proportion of parasites that are sequestered in the deep microvasculature. Although these sequestered parasitized erythrocytes are central to the pathophysiology of severe and complicated malaria, they are not reflected in the peripheral parasite count8. Total parasite biomass, which includes both the circulating and sequestered fractions, is better reflected by the plasma concentration of P. falciparum Histidine-Rich Protein 2 (PfHRP2)9, a 30 kD molecule released from the cytoplasm of falciparum-infected red blood cells, mainly during schizont rupture12. Plasma levels of PfHRP2 have diagnostic utility in differentiating between “true” severe malaria and other severe illnesses with incidental parasitaemia13.However, peripheral parasite densities are a poor measure of total parasite loads in PfHRP2 and the ratio of plasma PfHRP2 to the circulating parasite density are powerful quantitative phenotypic traits for case-only genetic association studies in severe malaria. We investigate associations between the malaria-protective polymorphisms identified in our earlier study7 and total parasite biomass estimated through plasma concentrations of PfHRP2, in a cohort of children admitted to hospital with a diagnosis of severe P. falciparum malaria in Kenya. The associations with parasite biomass help understand the mechanisms of protection for the studied polymorphisms.In this work we show that plasma PfHRP2 concentration were the admission platelet count (log transformed as previously described)13, the admission hemoglobin concentration, and parasite density. The overall distributions of parasite densities, plasma PfHRP2 concentrations, and ratio of PfHRP2 to parasites are summarized in Fig. After multiple imputation, we had data on a total of 2198 patients for our final analyses. The pattern of the missing data is shown in Supplementary Fig. HBB: reduction in geometric mean of 70% [95% CI 50–80]; ATP2B4: reduction of 30% [95% CI: 0–51]; ABO: reduction of 22% [95% CI: 5–36]). In comparison, four of the six major red blood cell polymorphisms  were associated with lower plasma PfHRP2 concentrations, while each had estimated larger effect sizes . For these four polymorphisms we compared the best fitting models of association in this case-only analysis to the best fitting models of association from previously reported case-control analyses. There were notable differences with previously published case–control studies regarding the best fitting models for these major polymorphisms. For rs334 (HbS) a heterozygous model of association was reported in the largest case–control study conducted to date, which included almost 12,000 cases and more than 17,000 controls, showing no apparent protective effect of homozygous sickle cell anemia (HbSS)5. However, in this case-only analysis of PfHRP2 as a quantitative trait, the best fitting model of association was additive (i.e. with greater protection in homozygotes than in heterozygotes). This offered strong evidence for rejecting the heterozygous model (likelihood ratio test comparing the heterozygous model with the two-parameter model: p = 10−5). For HBA1-2  the best fitting model was recessive, although the data were also consistent with an additive effect . The best fitting models for the rs186873296 mutation in FREM3 (Dantu), and the O blood group mutation in ABO (rs8176719) assumed additive and recessive models of association respectively, consistent with our previous study7. Polymorphisms in ATP2B4, G6PD, RPS6KL1, LOC727982, ARL14, LPHN2, IL10, CAND1, and GNAS were not associated with differences in baseline PfHRP2 and so no best model of association could be determined.As reported previously, only three polymorphisms were associated with lower parasite densities in cases , a highly significant association was seen with plasma PfHRP2 concentration . However, in the current analysis we found a trend towards higher plasma concentrations of PfHRP2 (p = 0.08), an effect in the opposite direction to that observed for the other malaria protective erythrocyte genetic polymorphisms. As a result, the association with the sequestration index (the PfHRP2/parasite ratio) was significant . Hemoglobin concentrations on admission were no lower in homozygotes than they were in wild type or heterozygotes, as would be expected if the higher plasma PfHRP2 concentrations were a consequence of a longer duration of illness . In contrast it decreased the evidence (p = 0.02 versus p = 0.003) for ATP2B4.We re-estimated case-control odds-ratios under a data-tilting framework for the five major red blood cell genetic polymorphisms evaluated using previously published probability weights based on the plasma ria Fig. 13,15. ThP. falciparum malaria, we show that the plasma PfHRP2 concentration is a useful phenotypic measure that can be used to validate genetic associations discovered through case–control studies. Consistent with previous reports, plasma PfHRP2 concentration is a better measure of disease severity than peripheral parasite density, and more accurately identifies true severe malaria. Our study shows that by providing a simple quantitative assessment of both disease severity and diagnostic accuracy, plasma PfHRP2 concentration can be a useful quantitative trait in genetic association studies.In this large cohort of children with severe 3 epidemiological support has accumulated for a number of candidate genes. However, in most cases the underlying mechanisms remain incompletely understood4. For example, several different hypotheses have been put forward for the best-established candidate, the sickle mutation in HBB. The mechanisms proposed fall into three broad categories. First, the early in vitro studies suggested that HbS containing red blood cells are less supportive of P. falciparum parasite growth and development, particularly under conditions of low oxygen tension18. Second, there is evidence that HbS affects the cytoadhesion of late-stage parasite-infected red cells to the vascular endothelium through reduced intracellular transport and expression of surface proteins, thereby attenuating the pathophysiological consequences of malaria21. Finally, it has been suggested that malaria parasite-infected HbS containing red blood cells may be removed more rapidly from the circulation through immunological mechanisms, particularly involving the spleen24. Our observation that both peripheral parasite densities and plasma PfHRP2 are considerably lower in children carrying the βs mutation is broadly compatible with all these three mechanisms. Similar mechanisms have been proposed for the malaria-protective effect of α-thalassemia yet, as in our current study, no associations have been observed with peripheral parasite densities in the majority of epidemiological studies4. Our novel finding that plasma PfHRP2 concentrations are substantially lower in children with α-thalassemia supports the conclusion that P. falciparum parasites are less able to thrive in carriers of this condition than they are in normal children. Finally, in a recent mechanistic study we found that red cells expressing the rare Dantu blood group are resistant to invasion by P. falciparum merozoites because they are less deformable than normal cells25. While in the current study this was not reflected by reduced parasite densities, we observed a 40% reduction in PfHRP2 concentration in Dantu children overall. Furthermore, the in vitro data showed a greater impact of Dantu for homozygous relative to heterozygous cells25, consistent with our in vivo data which suggest an additive association with plasma PfHRP2.During the seven decades since the “malaria hypothesis” was first proposedATP2B4 and the plasma PfHRP2 concentration and the sequestration index. The polymorphism involved, which results in red cell dehydration and raised concentrations of intracellular calcium26, was the third strongest protective signal in the largest multi-country case–control study of severe malaria conducted to date14. Nevertheless, in the current study we found that although the protective allele was associated with slightly lower parasite densities, yet plasma concentrations of PfHRP2 were higher, as was the sequestration index (the ratio of PfHRP2 to parasite density). If this corresponds to a true effect, it could have several possible explanations. One would be that parasite multiplication rates are lower in ATP2B4 homozygous patients, thus leading to higher plasma PfHRP2 concentrations relative to circulating parasites. However, this explanation would imply a longer illness course, which in theory should be associated with a greater degree of anemia, but this was not observed in the current study. Other potential explanations are that a greater amount of PfHRP2 is released into the plasma from the red cell at schizogony because less PfHRP2 is retained in the mutant cytoadherent ruptured ghost erythrocytes, or that the mutation results in enhanced PfHRP2 production. These hypotheses are testable in laboratory experiments.An unexpected finding of our study was the relationship between the polymorphism in Individual written informed consent, including consent to the storage and re-use of samples and data, was provided by the parents of all study participants. Ethical approval for the study was granted by the Kenya Medical Research Institute / National Ethical Review Committee in Nairobi, Kenya (SCC1192) and the Oxford Tropical Ethical Review Committee in Oxford, UK (020-06). Re-use of existing, appropriately anonymized, human data does not require ethical approval under the Oxford Tropical Research Ethics Committee regulations (OxTREC).7. Cases consisted of children aged <14 years who were admitted to the high dependency ward of Kilifi County Hospital between June 11th 1999 and June 12th 2008 with clinical features of severe falciparum malaria. Controls were infants aged 3–12 months who were born in the same study area as cases between 1st May 2006 and 30th April 2008 and were recruits to a cohort study investigating genetic susceptibility to a range of childhood diseases27. Controls were closely representative of cases in terms of sex, ethnic group, and geographic area of residence, but not age. The purpose of the controls was to provide background allele frequency estimates of the various genetic polymorphisms in the study population.The Kenyan severe malaria cohort has been described in detail previously28. Severe malaria was defined as a positive blood-film for P. falciparum parasites together with prostration (a Blantyre Coma Score of 3 or 4), cerebral malaria (a Blantyre Coma Score of <3), respiratory distress , severe anemia (hemoglobin concentration of <5 g/dL) or any other feature of severe malaria as defined by the World Health Organization29. Admission blood films were stained and examined by standard methods30 and parasite densities recorded as either the proportion of parasitized red blood cells in thin films (>1% of red cells parasitized) or based on the ratio of parasites to white blood cells in thick films for lower density infections. Parasite densities (the number of parasites/μl of whole blood) were then calculated with reference to full blood counts, if available, or if not, on the assumption of a white blood cell count of 8 × 103/μL or a red blood cell count of 5 × 106 /μL.Data and samples were collected through a clinical surveillance system that has been described in detail previouslyHBA1 and HBA2; -α3.7-thalassemia31) by PCR at the KEMRI-Wellcome Trust Laboratories, Kenya, as described previously33. Genotyping for a further 119 candidate SNPs was conducted at the Wellcome Centre for Human Genetics in Oxford, UK, by use of the Sequenom iPLEX MassARRAY platform, using DNA extracted from frozen samples of whole blood as described previously7. Plasma concentrations of PfHRP2 were batch-analyzed in Kenya by ELISA in admission samples stored at −80 °C using previously published methods34. We focused our analysis on 14 of the 15 previously associated genes: we excluded INPP4B because it tags the same nearby causal Dantu GYP mutation as FREM3, which is in stronger linkage disequilibrium35.Severe malaria cases were genotyped for the rs334 SNP and for the common African form of α-thalassemia (a 3.7 kb deletion in ABO (rs8176719), ATP2B4 (rs1541255), G6PD (rs1050828), HBA1-2 , HBB (rs334), FREM3/GYP (rs186873296), LPHN2 (rs72933304), CAND1 (rs10459266), RPS6KL1 (rs3742785), GNAS (rs8386), ARL14 (rs75731597), CD40LG (rs3092945), IL10 (rs1800890), and LOC727982 (rs1371478) on three quantitative parasite-specific traits: (i) peripheral parasite density (the number of circulating parasites per μL); (ii) plasma PfHRP2 concentration (ng/mL), a proxy measure of the total parasite biomass; and (iii) the ratio of the plasma PfHRP2 concentration to the circulating parasite density (ng/parasite), which is proportional to the previously reported sequestration index9. Supplementary Table PfHRP2 by a constant term which is an estimate of the multiplication rate over the previous life cycle9. The PfHRP2 to parasite ratio is simpler to interpret as it does not make any assumptions about multiplication rates , an issue that is particularly pertinent in the context of malaria-protective genes. For all three endpoints we used a log10 transformation as the parasite density and plasma PfHRP2 concentration are both approximately log-normally distributed. The log10PfHRP2 to parasite ratio is thus expressed as the difference between the two log10 transformed variables. To aid the comparison of effect sizes across the three quantitative traits we standardized each dependent variable to obtain distributions with mean 0 and standard deviation 1. To account for parasite densities that were below the lower limit of detection by thick film microscopy (~50 parasites/μL), we used a log10(x + 50) transformation for the peripheral parasite density. We excluded non-measurable PfHRP2 concentrations (22/1522 samples) as these could represent HRP2/3 parasite gene deletions or assay errors. Out of 2220 genotyped severe malaria patients, measurable PfHRP2 concentrations were available for 1500 patients and parasite densities were available for 2184 patients. In addition, we included patient data on platelet counts, admission hemoglobin and other key admission variables relating to patient severity and demographics. To increase power, we performed multiple imputation using random forests for the patients with missing PfHRP2 concentrations and missing parasite densities, excluding patients who had missing PfHRP2 concentration, parasite count and platelet count data (n = 22 patients). Ten imputed datasets were generated (R package missForest). Two sensitivity analyses were performed: first, we included additional covariate adjustment for hemoglobin, age, and bacteremia; second, we estimated effect sizes for the same association models using only patients with non-missing phenotype data .We explored the effects of polymorphisms in ABO rs876719, ATPfHRP2-based traits including general two-parameter models, additive, heterozygous and recessive models, and determined the best fitting models from the model likelihoods. The latter three models of association were then compared against the general two-parameter model using a likelihood ratio test with 1 degree of freedom. A p-value threshold of 0.05 was used to reject an association model. All p-values are reported without correction for multiple testing. Parameter estimates for each imputed dataset were combined using Rubin’s rules (R package mitools) and are given in Supplementary Tables PfHRP2 for the severe malaria cases, we estimated case-control odds-ratios for protection against severe malaria, using probabilistic data-tilting to down-weight patients who likely did not have severe malaria, using a previously reported model based on PfHRP2 and platelet counts13. Tests for Hardy–Weinberg equilibrium were reported in our earlier study7 for all 14 SNPs, with no significant departures from expected genotype frequencies. All analyses were done in R v4.0.2.Quantitative trait associations were assessed under linear models with Gaussian errors. All models were adjusted for self-reported ethnicity. We fitted a range of association models to the parasite density and plasma Further information on research design is available in the Supplementary InformationPeer Review FileReporting Summary


Microscopic examination found five cases of P. falciparum + P. vivax infection, while PCR found 346 cases. All microscopic examinations turned negative in the post-DHP-therapy group. Conversely, PCR result from the same group yielded 29 negative results. Overall, our study showed that microscopic examination and PCR generated different results in detecting Plasmodium species, especially in patients with mixed infection and in patients who recently underwent DHP therapy.Microscopic examination is the backbone of malaria diagnosis and treatment evaluation in Indonesia. This test has limited ability to detect malaria at low parasite density. Inversely, nested polymerase chain reaction (PCR) can detect parasites at a density below the microscopic examination’s detection limit. The objective of this study is to compare microscopic and PCR results when being used to identify malaria in suspected patients and patients who underwent dihydroartemisinin–piperaquine (DHP) therapy in the last 3–8 weeks with or without symptoms in Sumba Barat Daya, Nusa Tenggara Timur, Indonesia. Recruitment was conducted between April 2019 and February 2020. Blood samples were then taken for microscopic and PCR examinations. Participants ( In recent years, malaria cases in Indonesia have been showing a declining trend. In fact, more than half of the districts in this country were free from malaria in 2017 . For insAs a primary health center (PHC) in Sumba Barat Daya District, Kori PHC reported 1,343 cases of malaria in 2020, rising sharply from 487 cases in 2019 [P. falciparum alone [P. malariae mono-infection, DHP alone for 3 days was given [P. vivax or P. ovale infection, whether it was mono or mixed infection, a 14-day course of primaquine was given in addition to a 3-day course of DHP [Indonesian national guideline recommends dihydroartemisinine–piperaquine  fixed-dose combination with or without 0.25 mg/kg body weight of primaquine to treat uncomplicated malaria ,7,8. HowMicroscopic examination is the backbone of malaria diagnosis in Indonesian PHCs. This method can distinguish parasite species and stages, quantify parasite density, and is inexpensive . In endePlasmodium sp. among the residents of Kodi Utara Subdistrict, Sumba Barat Daya District. By doing this, we were able to identify cases of asymptomatic malaria in this population and identify the Plasmodium species infecting these patients.This cross-sectional study aimed to compare the results from microscopy and PCR examination when being used to detect This study utilized a cross-sectional design. Participants were divided into 3 groups: suspected malaria, post-DHP therapy with fever, and post-DHP therapy without fever. For each participant, blood samples were taken for microscopic and PCR examination. The results of both diagnostic modalities were then compared to each other. n = 409) who fulfilled inclusion criteria were divided into three groups: those who were suspected of malaria , post DHP therapy with fever , and post-DHP therapy without fever .The population of this study is the resident of Kodi Utara Subdistrict, Sumba barat Daya District at Sumba Island, NTT. Participants were recruited from patients that visited Kori PHC as well as residents who recently underwent DHP therapy that were followed up by a local malaria cadre. Recruitment was conducted between April 2019 and February 2020. The inclusion criteria for this study are local residents of Kodi Utara Subdistricts who were either suspected of malaria or just recently underwent DHP therapy with or without primaquine in the last 3–8 weeks. In addition, we excluded patients who were suspected of suffering from severe malaria, pregnant women, and resident who declined to participate. Participants , Yogyakarta, Indonesia.Thin and thick slides of peripheral blood were made after collection and allowed to air dry. Slides were stained with 3% Giemsa solution for 45 min at room temperature . Slides P. falciparum, P. vivax, P. ovale, and P. malariae were amplified as described previously with slight modifications [The DNA was extracted from venous blood collected in an EDTA tube using a commercial kit (Geneaid Kit) and stored at −20 °C. Using the 18s ribosomal RNA  as a refications . The volThe results of microscopic examination and PCR were analyzed by McNemar’s test to assess whether the proportions differed from repeated measurements in one sample.Plasmodium sp. [Asymptomatic malaria was defined as an asymptomatic individual whose microscopic and/or molecular examination results show the presence of dium sp. ,24. Micrn = 409) were divided into three groups: suspected of malaria , post-DHP therapy with fever , and post-DHP therapy without fever . Characteristics of these participants are shown in Participants , P. vivax , and P. falciparum + P. vivax . Meanwhile, samples from post-DHP therapy patients without fever showed the presence of P. vivax  and P. falciparum + P. vivax , even though the patients were asymptomatic during the presentation. P. ovale were not identified in any sample by both microscopic and PCR examination. Finally, only 29 (7.1%) samples were found to contain no parasite by both microscopic and PCR examination.Plasmodium species were not found by microscopic examination in patients that underwent DHP therapy, regardless of the presence of fever. However, PCR examination showed different results. Samples from post-DHP therapy patients with fever showed submicroscopic parasitemia that contained P. falciparum was the leading cause of illness . However, PCR results showed that mixed infection by P. falciparum + P. vivax was instead the leading cause of illness in this group . Meanwhile, microscopic examination seemed to miss six cases of mixed P. falciparum + P. vivax + P. malariae detected by PCR. Instead, these cases were identified as mono infection by P. malariae.The microscopic examination result from the suspected malaria group suggested that mono infection by P. falciparum mono infections, while nested PCR only found 11 (p < 0.001). P. vivax mono infection was found in 34 samples by microscopic examination and in 16 samples by nested PCR (p < 0.001). Microscopic examination found mixed P. falciparum + P. vivax infection in five samples, but nested PCR results showed 346 samples were infected with both species (p < 0.001). The results of the microscopic examination showed mono infection by P. malariae in seven samples, but nested PCR showed only one sample was infected P. malariae alone (p = 0.031). Six cases of mixed infection by P. falciparum + P. vivax + P. malariae were found by PCR. However, the microscopic examination did not find any samples infected by these groups of pathogens. Most microscopic examinations in this study yielded negative results (n = 235). However, only 29 samples examined by nested PCR returned negative results (p < 0.001).McNemar test showed a significant difference between the result of microscopic examination and nested PCR. Overall, microscopic examination found 128 Comparison between the results of microscopic examination and nested PCR when participants visited the Kori PHC are shown in The result of our study showed that microscopic examination and PCR have visibly different results when being used to detect parasitemia. This is especially true in cases of mixed infection and in groups of patients who recently underwent DHP therapy.In our study, microscopic examination was only able to identify five mixed infections, while PCR found 351 mixed infections. The limitation of microscopic examination in detecting mixed infection has been well-documented ,17,18. AP. vivax, with the majority of them occurring in the form of mixed P. falciparum + P. vivax infection. This is in contrast to the local government report, which mentioned that 68% of malaria cases in NTT province—where this study was conducted—was caused by P. falciparum, with P. vivax contributing to only 26% of the case [P. vivax distribution is difficult, especially using microscopic examination. This is because P. vivax infection tends to be asymptomatic and has low parasite density, which may lead to false negative microscopic examination result [The dominant species found in our study is the case . Howevern result .P. vivax (33.9%) is the most dominant species in this region, with P. falciparum found in only 24.8% of cases [P. falciparum and P. vivax was found in 43.1% and 39.6% of positive samples, respectively [P. vivax mono-infection in 33 out of 35 positive samples [Plasmodium species epidemiology in Indonesia using molecular methods is a path worth exploring.Owing to its ability to detect parasites at lower parasite density than microscopic examination, molecular methods such as PCR can be used as an alternative epidemiological surveillance method. Several Indonesian studies have employed this strategy, with varying results. For example, surveillance conducted in North Sumatra province in 2015 revealed that of cases . A similectively . A small samples . UnfortuP. vivax at community level is challenging. In fact, Adekulne, et al. estimated that more than 70% of P. vivax infections in Thailand and Papua New Guinea arise from hypnozoite reactivation [Due to its ability to form dormant hypnozoites, management of tivation . The reltivation . This sttivation . P. vivax. The combination of DHP with or without primaquine is the mainstay of therapy for uncomplicated malaria in Indonesia [P. falciparum infection, primaquine was given as a single dose, while a 14-day daily dose of primaquine was given for P. vivax infection [P. vivax infections were missed by microscopic examination. Consequently, these patients did not receive proper primaquine dosing. Given that DHP could not eliminate hypnozoite [P. vivax. This suspicion is supported by our findings in the post-DHP therapy group.The frequency of relapse can be suppressed by administering anti-malaria that targets hypnozoite stage of ndonesia . For P. nfection . In our pnozoite , we stroP. falciparum + P. vivax infection despite negative microscopic examination results. This suggests that recent administration of DHP likely reduced—but did not eliminate—Plasmodium sp. in patient’s blood to below the detection threshold of microscopy. Regardless, delayed conversion of PCR results following anti-malarial therapy was also observed in other studies. For instance, Vafa Homann et al. [In the post-DHP therapy group, most patients received positive PCR results for mixed n et al.  found pon et al. .P. falciparum DNA from a patient’s circulation following initiation of therapy [P. falciparum remains positive for at least 42 days following treatment initiation [P. vivax infections missed by microscopy, we suspect that it is unlikely that positive PCR results for this species in the post-DHP therapy group came from the remnant of the dead parasite.Interpretation of PCR results following the administration of therapy must be made cautiously, as detection of genetic material from the dead parasite is possible. A mice study suggested that parasite DNA was rapidly cleared from the mice’s circulation following parasite killing . However therapy . Anotheritiation . The stuitiation . Due to Regardless, we acknowledge that this study has several limitations that may limit its interpretation. For instance, due to its cross-sectional design, interpreting the temporality in the relationship between PCR and microscopic examination results is not feasible. Furthermore, we could not quantify the parasite DNA recovered from our sample due to the type of PCR that we used. Taken together, these limitations prevent us from confirming the reason behind the discrepancy between PCR and microscopy results in our study.P. vivax infection that was undiagnosed by microscopic examination. This can be done by utilizing the qPCR technique to see whether there is a fall in the amount of parasite DNA over time following the initiation of proper treatment. If undiagnosed P. vivax is truly the cause, then there is an urgent need to reform the detection method in the community to eliminate malaria from the region.We believe that it is important to verify whether the positive PCR in the post-DHP therapy group truly stem from the detection of Microscopic examination and nested PCR have noticeably different results when being used to detect Plasmodium species. The difference in the result yielded by the two diagnostic modalities is especially apparent in cases of mixed infection and in groups of patients who recently underwent DHP therapy. Considering the limitation of microscopic examination and the outcome of our study, we believe that an evaluation of the malaria testing policy is needed.


Malaria is a parasitic infection that is a great public health concern and is responsible for high mortality rates worldwide. Different strategies have been employed to improve disease control, demonstrating the ineffectiveness of controlling vectors, and parasite resistance to antimalarial drugs requires the development of an effective preventive vaccine. There are countless challenges to the development of such a vaccine directly related to the parasite’s complex life cycle. After more than four decades of basic research and clinical trials, the World Health Organization (WHO) has recommended the pre-erythrocytic Plasmodium falciparum  malaria vaccine for widespread use among children living in malaria-endemic areas. However, there is a consensus that major improvements are needed to develop a vaccine with a greater epidemiological impact in endemic areas. This review discusses novel strategies for malaria vaccine design taking the target stages within the parasite cycle into account. The design of the multi-component vaccine shows considerable potential, especially as it involves transmission-blocking vaccines (TBVs) that eliminate the parasite’s replication towards sporozoite stage parasites during a blood meal of female anopheline mosquitoes. Significant improvements have been made but additional efforts to achieve an efficient vaccine are required to improve control measures. Different strategies have been employed, thus demonstrating the ineffectiveness in controlling vectors, and parasite resistance to antimalarial drugs requires the development of a preventive vaccine. Despite having a vaccine in an advanced stage of development, such as the RTS, S malaria vaccine, the search for an effective vaccine against malaria is far from over. This review discusses novel strategies for malaria vaccine design taking into account the target stages within the parasite’s life cycle. Plasmodium spp. parasites, transmitted by bites from infected female Anopheles mosquitoes. According to the World Health Organization (WHO), globally, there were an estimated 247 million malaria cases in 2021 in 84 malaria endemic countries (including the territory of French Guiana), an increase from 245 million in 2020, with most of this increase coming from countries in the WHO African Region [Malaria is an infectious disease caused by n Region . CurrentPlasmodium vivax, Plasmodium falciparum, Plasmodium malariae, and Plasmodium ovale are among the species that commonly affect humans. P. falciparum is responsible for the most severe form of disease and death and is more prevalent in Africa. The second most common species is P. vivax, found in South and Southeast Asia, Central and South America, and some countries in Europe and North Africa [Plasmodium antigens or immunogenic fragments have been reported . Due to the complexity of the Plasmodium biological cycle, most vaccines reported against malaria have more than one target antigen. This review reports the patent landscape for malaria vaccines using different target stages.h Africa . The cych Africa . After fh Africa ,6. Interh Africa . Severalhttps://worldwide.espacenet.com (accessed on 4 May 2022)), United States Patent and Trademark Office (USPTO—https://www.uspto.gov/patents-application-process (accessed on 4 May 2022)), United States Latin America (LATIPAT—https://lp.espacenet.com/ (accessed on 4 May 2022)), and Patentscope -Search International and National Patent Collections (WIPO—https://patentscope.wipo.int (accessed on 4 May 2022)). The keywords used were: “malaria vaccine” AND “pre-erythrocytic” OR “sexual” OR “blood”. The titles and abstracts from pre-selected articles were read and evaluated by two independent researchers.The following patent databases were consulted: European Patent Office  vaccine (trade name Mosquirix) [P. falciparum infection [The most advanced candidate to-date is the pre-erythrocytic squirix) . The mosnfection , (ii) sanfection , and , plateaued after two immunizations in a clinical trial of the PfSPZ vaccine. It was determined that recall responses were inhibited by antibody feedback, potentially through epitope masking of the immunodominant PfCSP repeat region [There is general consensus that generating enough antibodies to block infection is a key challenge for induction protection in malaria vaccines . Another vaccine . The autt region ,33. McNat region ,35. ThesFinally, there is a pre-erythrocytic malaria vaccine candidate R21/Matrix-M that was developed at the University of Oxford  and is currently manufactured by the Serum Institute of India . The ongoing safety, immunogenicity, and efficacy of R21/Matrix-M, and the number of malaria cases averted by this vaccine over 2 years of follow-up, following administration of the first booster dose have been reported .P. falciparum and the limitations imposed by animal models. Scientists look forward to the discovery of new antigens and the improvement of platforms to expand the existing portfolio of vaccine candidates.Despite the advances, there is still a strong desire to find a highly effective vaccine against malaria. The question remains as to how effective and safe malaria vaccines can work to benefit those communities ravaged by malaria. Furthermore, there are challenges yet to be overcome regarding the development of such a vaccine, such as the extremely complex genome of + T cells play a critical role in the response to the pre-erythrocytic stage of malaria [+ T cells are recognized as the primary effector cells against the pre-erythrocytic stages, as observed in non-human primates [Sporozoites travel through the blood in the pre-erythrocytic stage and infect hepatocytes which then undergo schizogony, the multiplication stage that precedes the invasion of red blood cells . The mai malaria ,43. Furtprimates . P. falciparum genome [Plasmodium genome allowed the development of live parasites attenuated by more precise and better-defined genetic manipulations. It was the strategy used in the patent US5112749A  are considered as essential for the early development of the liver stage [Plasmodium organisms, the sporozoites UIS3 (-) and UIS4 (-), which infect hepatocytes, but are no longer able to establish infections in the blood stage and, therefore, do not lead to disease.Gardner et al. sequenced the m genome . The ava5112749A , referri38580 A1  also pronvention , which rer stage . This teP. falciparum. It has been shown that radiation required to generate an effective attenuated sporozoite must be precisely adjusted to meet minimum requirements [Plasmodium sporozoites have been reported [P. falciparum sporozoites presents safety concerns that cannot be ignored [Despite these promising results, there are some disadvantages in using attenuated microorganisms as represented by need for the attenuation of irements . Moreoveirements . Likewisreported . Since a ignored .Plasmodium parasites as a non-pathogenic vector for human immunization, was presented by the AU2013250814B2 invention  was used and shown to maintain the primary characteristics of its wild-type counterpart while triggering a specific protective immune response against the P. falciparum challenge. This strategy opens other avenues for the design and production of additional vaccine candidates based on the same principle. While the development of a live vaccine could raise concerns regarding safety requirements, in addition to scale-up in the vaccine production, the EP1544211A1 invention . This protein is highly antigenic and the prevalence of antibodies in individuals living in endemic areas is extremely high (roughly 90%). Immunization with LSA-5 induced protection against both challenges of P. yoelii (in mice) and P. falciparum (in Aotus monkeys). The results suggest that LSA-5 could be an important antigen candidate for an anti-malaria subunit pre-erythrocytic vaccine.An alternative strategy for the development of a malaria vaccine, based on the use of rodent nvention . It has nvention  describeP. falciparum involves a complex cascade of protein-protein interactions between the parasite’s ligands and the host’s receptors [P. falciparum family (PfRh) are involved in binding and initiating invasive merozoite entry into erythrocytes [Plasmodium parasite blood-stage genetic strains. However, functional RH5 is only expressed in eukaryotic systems and new approaches are needed to stabilize the immunogen [Plasmodium parasites. The inventors of US20140186402A1  with a modified gene to prevent infection in the host’s red blood cells. The P. falciparum depends on the acquisition of purines from the host for its survival in human erythrocytes [In the US20080026010 invention , the authrocytes . Purine hrocytes . The invP. falciparum asexual stages. The mixture of peptide compounds was injected into Aotus trivirgatus monkeys, inducing high antibody titers against the peptides and reacting with the P. falciparum parasite. The Colombian A. trivirgatus monkeys were immunized and challenged intravenously with blood cells infected with 5 × 106 P. falciparum parasites obtained from an infected A. trivirgatus donor monkey. No parasites were detected in blood smear samples up to 90 days after challenge. The vaccine was also tested on human volunteers who were vaccinated two or three times with the synthetic protein copolymer. The volunteers were exposed to an experimental intravenous inoculation of red blood cells infected with one million fresh live ringed P. falciparum particles, resistant to grade chloroquine and with complete sensitivity to sulfadoxine and pyrimethamine, as are most wild Colombian strains. The composition was shown to induce complete and self-limited protection; three of the five vaccinated volunteers had mild infections with continual decrease in parasite count and full recovery on day 21. This synthetic hybrid protein, referred to as SPf 66, provides the first safe synthetic vaccine against the asexual stages. Moreover, a recombinant protein SE36, with 47 kDa, expressed in E. coli, was described in the invention US20040137512 as a highly effective formulation to prevent the P. falciparum growth in the blood considering human IgG3 antibodies are able to specifically bind to SE36 protein, thus blocking parasite growth.Alternatively, the use of a synthetic malaria vaccine instead of live parasites is described in the patent US4957738 . This inPlasmodium parasites by the Anopheles vector. As a result, the spread of malaria is expected to decline with reduction in the disease. The specific antibodies generated in the human host are passively ingested together with parasites when mosquitoes take a blood meal and will bind to the parasites, thereby preventing the progression of their sporogonic development [Another type of approach targets vector control and parasite transmission strategies through the development of transmission blocking vaccines (TBVs) . In thiselopment . Once inelopment . Biologielopment . P. falciparum 48/45 (Pfs48/45), was described as expressed by gametocytes [P. falciparum. The WO2010036293A1 patent  or P. vivax P47 (Pfs47) surface antigens. The inventors proposed these proteins for blocking or reducing the infection by P. falciparum or P. vivax in A. gambiae or other anopheline mosquitoes and, thus, preventing the parasite transmission. In US20150191518A1 . The inventors discovered that the emerging gametes of P. falciparum bind the complement regulator factor H (FH) following transmission to the mosquito to protect against the complement-mediated lysis by the blood meal [P. falciparum, resulting in the destruction of the malaria parasite by the human complement of the blood meal. Anopheles stephensi were artificially fed with neutral mouse antiserum and mouse Anti-PfGAP50 and the presence of the anti-PfGAP50 antibody reduced transmission rates by 68%.The specific antigenic target, the surface antigen of etocytes  on the setocytes , and theetocytes . This ap1 patent  also des191518A1 , the autMalaria transmission-blocking vaccines are advancing in clinical trials ,64 and sNotably, one of the major challenges to developing a malaria vaccine is the parasite’s complex life cycle and its various stages of development. The first barrier that the malaria vaccine needs to control is the pre-erythrocytic phase, which requires protection against the infectious (sporozoite) form injected by mosquito and inhibits the development of parasites in the liver . HoweverPlasmodium infection was described in invention EP2923709A1  or fragments thereof; and the iii sexual stage antigens include the ookinete antigen Pfs25 and/or the gamete/gametocyte surface protein Pf230C0 or variants or fragments thereof. The combination of recombinant proteins and fusion proteins outlined in this patent trigger a protective immunity that blocks infection, in addition to preventing the spread of the disease and interrupting the transmission of parasites. Rabbits were immunized and antibodies to rabbit antisera were purified by protein A chromatography. Sporozoite binding/invasion inhibition assays were performed to assess the ability of antisera directed against P. falciparum antigens to block the attachment and invasion of P. falciparum NF54 sporozoites to liver cells. The result was a 30% inhibition. Furthermore, there was an 80% inhibition in the GIA assay. Finally, membrane feeding assays were performed to assess the ability of antisera directed against P. falciparum antigens to block the transmission of P. falciparum NF54 from human to mosquito, and the transmission-blocking rates were between 80% and 100%. Using a similar approach, the authors of the invention CA2910322A1 , merozoite surface protein Pf Msp1-19, and peptides derived from Pf Rh5 and Pocs 25 antigen (EP2992895). Furthermore, the WO2017142843 invention (Plasmodium that induce sterile protection (100%) in mice against an infectious sporozoite challenge from P. yoelii.The idea to provide an additional immune response to the first, second, and third barriers against the 923709A1 . This te910322A1  proposednvention  providesPlasmodium falciparum T-cell specific circumsporozoite protein (CSP) responses, expansion in the amplitude of the αCSP IgG response, and increased inhibition of sporozoite invasion in hepatocytes with CSP-p (Man-TLR7), as compared with CSP, formulated with MPLA/QS-21 -loaded liposomes, the adjuvant used in the most clinically advanced malaria vaccine [The new pandemic of coronavirus disease COVID-19) caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) presents new challenges to public health programs worldwide . In coun caused b vaccine . E. coli cell-free systems [Plasmodium surface proteins, such as CSP, has the potential to influence the humoral response to a vaccine, such as RTS, S. For example, glycosylation may increase glycoprotein endocytosis by antigen-presenting cells and protect against excessive proteolysis of glycopeptides within the endolysosome by steric occlusion [Another recent advance has renewed prospects for the accelerated discovery of key malaria antigens, optimization of protein synthesis platforms through high-throughput immune screening approaches, reverse vaccinology, structural immunogen design, lymphocyte repertoire sequencing, and the use of artificial intelligence . The whe systems . The dis systems . Post-tr systems . The precclusion .Immuno-profiling and reverse vaccinology approaches are essential to accelerating research and the development of new vaccine targets. A key message learned from these pioneering approaches is that it is crucial to select an expression system with the ability to produce many correctly folded recombinant malaria proteins without artificial glycosylation . The devPlasmodium is required to obtain a highly effective malaria vaccine. In this sense, the multi-component vaccine strategy based on blocking the Plasmodium infection shows great potential since it considers acting at three levels: (i) the pre-erythrocytic stage, (ii) the blood stage, and (iii) the sexual stage. As expected, the integrative approach using distinct Plasmodium antigens to limit each parasite’s life cycle could increase the malaria vaccine’s protection rate. The use of new technologies, such as nanoparticle glycoengineering, could play an important role in the development of emerging vaccines, such as a malaria vaccine. Concerns over the restricted experimental models used to analyze the anti-malaria vaccine candidates limit the global effort to develop an effective formulation that can be produced on a large scale. Considerable progress has been made in recent years in developing an effective vaccine against malaria. However, with the emergence of COVID-19 and efforts aimed at containing the pandemic, this cannot be allowed to restrict further advances toward making an effective vaccine commercially available.As this review has made clear, the complexity of the malaria parasite cycle makes the development of a malaria vaccine a very difficult task. The parasite manages to circumvent the immune defenses by continuing the cycle. An approach to limit the immune system’s evasion mechanism by the


Genomic epidemiology has guided research and policy for various viral pathogens, and there has been a parallel effort towards using genomic epidemiology to combat diseases that are caused by eukaryotic pathogens, such as the malaria parasite. However, the central concept of viral genomic epidemiology, namely that of measurably mutating pathogens, does not apply easily to sexually recombining parasites. Here we introduce the related but different concept of measurably recombining malaria parasites to promote convergence around a unifying theoretical framework for malaria genomic epidemiology. Akin to viral phylodynamics, we anticipate that an inferential framework developed around recombination will help guide practical research, and thus realise the full public health potential of genomic epidemiology for malaria parasites and other sexually recombining pathogens. The public health value of malaria genomic epidemiology see  has beenGenomic epidemiology relies on the concept of measurably evolving pathogens , 12. A phttps://nextstrain.org/pathogens. In general, malaria parasites are not counted among them, partly because the coherent inferential framework that applies to fast-mutating RNA viruses (phylodynamics) does not apply readily to malaria parasites since they sexually recombine.The conventional definition of a measurably evolving pathogen assumes genetic differences are generated by mutation , 12. PatPlasmodium, the concepts apply to a broader range of sexually recombining pathogens.In this article we compare the genetic consequences of recombination versus mutation in the context of malaria genomic epidemiology, examine methodological gaps, and propose an approach towards an unifying inferential framework, something akin to phylodynamics in viral genomic epidemiology. Although we focus on δ, can be modelled simply as linear function of time t, the rate of mutation per locus per time μ, and the number of loci l : δ = μlt [Plasmodium falciparum and Plasmodium vivax, the two malaria parasite species most frequently responsible for human malaria [Both mutation and recombination generate genetic variation , 11: mut δ = μlt . Sexual  malaria .P. falciparum has a 23 megabase nuclear genome [−10 mutations per base pair per asexual generation (48 hours) [Malaria parasites are single-celled and, throughout the human stage of their lifecycle, haploid. Compared with viral pathogens, they have larger genomes but slower mutation rates: r genome  and a SN8 hours) .P. falciparum genomic epidemiology [P. falciparum [Although this process generates many mutations - given the vast amount of parasites within a single malaria infection - the majority of those mutations occur singularly and are purged . A well-emiology . Among aemiology . That vaemiology . Thus, wlciparum , it is tlciparum .While malaria parasites might accrue a small number of non-purged mutations over the course of one lifecycle, 50% of the genome is expected to differ if recombination with an unrelated parasite occurs. This means that recombination has greater potential to generate measurable variation on epidemiologically relevant timescales. This potential has been demonstrated by various studies e.g. , 20, 21), 2120, 2P. falciparum gametes are estimated to crossover with probability 7.4 × 10−7 per base pair [P. falciparum has 14. This means that after recombination between unrelated parasites, we expect offspring to be 50% related to their parents with, on average, one contiguous IBD segment per chromosome. Even without crossovers, sexual reproduction can generate variation because offspring inherit a random combination of their parental chromosomes.Recombination is obligate in the malaria parasite lifecycle. Human blood stage parasites differentiate into gametocytes that are imbibed by the mosquito, where they differentiate promptly into gametes, and pair to sexually recombine approximately three hours after ingestion , each paase pair . This imAlthough recombination is obligate, it is not always effective. Malaria parasites can self, i.e. genetically identical parasites can recombine, in which case recombination is ineffective. Selfing is inevitable when a mosquito feeds on a single monoclonal infection. Otherwise, selfing, inbreeding and/or outcrossing can occur, where inbreeding refers to partially effective recombination between related parasites, outbreeding refers to fully effective recombination between unrelated parasites, and the occurrence of one or more events depends on the number of parasite pairs that recombine. The extent of effectiveness depends principally on the composition of multiclonal human infections on which mosquitoes feeds .A multiclonal human infection can be generated in two, non-mutually exclusive ways: by a single mosquito bite transmitting genetically distinct parasites (cotransmission) and/or by several mosquito bites (superinfection), e.g. Both cotransmission and superinfection are expected to increase with transmission intensity, thereby increasing the overall prevalence of multiclonal infections (as has been observed inversely ) and theP. vivax, where MOIs can reflect present or past innoculations and thus are generally higher that those of P. falciparum [Observations from field studies testify to the complexity of this system, using descriptive statistics of parasite genetic data as proxy indicators. Generally, high estimates of diversity and average multiplicity of infection (MOI) suggest high transmission, while evidence of prevalent clonal clusters and monoclonal infections suggest low transmission . Howeverlciparum .P. vivax population diversity and average MOI has been observed together with significant linkage disequilibrium  [P. falciparum population diversity and prevalent monoclonal infections has been observed together with low LD [P. falciparum inbreeding persists [P. falciparum multiclonal infections contain highly related parasites [For either species, what these processes collectively mean for the effectiveness of recombination is unclear: in low transmission settings, evidence of high ination) , 37; whih low LD . In highpersists , 45, conarasites , 26, 47.To summarise, effective recombination has greater potential than mutation to generate variation that is measurable on an epidemiological scale, but, unlike mutation, its effectiveness is inextricably linked to the epidemiological context in a complicated way . AlthougFor the practical application of pathogen genomic epidemiology, data should be used to infer parameters of epidemiological interest under a cohesive statistical model that links the processes that generate the genetic data to epidemiological ones Descriptive analyses in malaria genomic epidemiology are related to those across malaria genomics more generally see . They gehttps://mrc-ide.github.io/SIMPLEGEN/). Agent-based models linked to genomic processes have been used to estimate R0 and changes in transmission intensity [Because of recombination, phylodynamic frameworks cannot be applied directly to malaria  and an ntensity , 4, to intensity , 55, to ntensity , and to ntensity . These mThe ultimate unifying inferential framework for malaria genomic epidemiology would centre around ancestral recombination graphs (ARGs), in the same way phylodynamics centers around phylogenetic trees. In the case of malaria, the mating system would link host-level parameters of epidemiological interest to the parasite genomes that feature in the ARG.An ARG is a graph that links DNA sequences by both mutation and effective recombination. It can also be viewed as a sequence of phylogenetic trees, one tree for each locus along the genome, where trees from one loci to the next are transformed if effective recombination events occur between the loci . It is aThe locations of effective recombination events in a malaria parasite ARG and thus the effectiveness of recombination are governed by the parasite mating system: branches are sampled uniformly at random among branches allocated to different broods when non-brood mating occurs, branches are sampled among those allocated to the same brood when brood mating occurs; otherwise, when recombination is not effective, branches are propagated from one generation to the next. How to model the malaria parasite mating system is a difficult open question but models from population ecology provide some inspiration. The use of IBD, though relatively new to malaria genomic epidemiology, is not new to population ecological studies of eukaryotes, where sexual recombination is the primary source of genomic variation. For example, Close Kin Mark Recapture, a method recently developed to estimate time-series of adult population size and survival of fish or other species , such asMalaria genomic epidemiology is an exciting field of research which has proven useful for informing malaria surveillance and in which interest is growing among malaria control programs and policy makers. However, it is largely dominated by descriptive genomic data analysis that are disconnected from routine epidemiological analyses and are frequently retrospective. Although insightful, these types of analyses lack a clear common framework and are limited to speculative interpretation of the underlying transmission dynamics. We introduce the concept of measurably recombining malaria parasites in the hope that it will encourage development around a unifying inferential framework under which models can be developed and thus used for hypothesis-driven analyses and statistically robust prospective study design. This is by no means an easy task see  but ongo


Malaria and filariasis are significant vector-borne diseases that are co-endemic in the same human populations. This study aims to collate the evidence, probability, and characteristics of malaria and filariasis co-infections in participants among studies reporting the co-occurrence of both diseases.We searched for potentially relevant articles reporting the co-occurrence of malaria and filariasis in five electronic databases  from inception to May 22, 2022. We estimated the pooled prevalence and probability of malaria and filariasis co-infections among study participants using random-effects meta-analyses and synthesized the characteristics of patients with co-infections narratively.Wuchereria bancrofti, malaria and Loa loa, malaria and Mansonella perstans co-infections were 0.7%, 1.2%, and 1.0%, respectively. The meta-analysis results showed that the co-infections between two parasites occurred by probability (P = 0.001). Patients with co-infections were at increased risk of having an enlarged spleen, a lower rate of severe anemia, lower parasite density, and more asymptomatic clinical status. Patients with co-infections had decreased levels of C-X-C motif chemokine 5, tumor necrosis factor–α, interleukin-4, c4 complement, and interleukin-10. In addition, patients with co-infections had a lower interleukin-10/tumor necrosis factor–α ratio and higher interleukin-10/interleukin-6 ratio.We identified 951 articles, 24 of which  met eligibility criteria and were included in the systematic review. Results of the meta-analysis showed a pooled prevalence of malaria and filariasis co-infections among participants of 11%. The prevalence of co-infections was 2.3% in Africa, 0.2% in Asia, and 1.6% in South America. The pooled prevalences of malaria and The present study showed that the prevalence of malaria and filariasis co-infections was low and varied between geographical areas in the selected articles. Co-infections tended to occur with a low probability. Further studies investigating the outcomes and characteristics of co-infections are needed. Anopheles mosquitoes infected with the protozoan genus Plasmodium species. Filariasis is caused by the infections of filarial nematode diseases, including lymphatic filariasis, mansonellosis, and loiasis through the bite of Anopheles and other vectors. We searched and synthesized the literatures reporting the co-occurrence of malaria and filariasis. We found that the overall prevalence of malaria and filariasis co-infections among participants of 11%. The prevalence of co-infections was 2.3% in Africa, 0.2% in Asia, and 1.6% in South America. The overall prevalences of malaria and lymphatic filariasis, malaria and loiasis, malaria and mansonellosis co-infections were 0.7%, 1.2%, and 1.0%, respectively. The co-infections between two parasites occurred by probability. Patients with co-infections of both diseases were at increased risk of having an enlarged spleen, a lower rate of severe anemia, lower parasite density, and more asymptomatic clinical status. In addition, patients with co-infections showed alterations of cytokine and chemokine levels such as C-X-C motif chemokine 5, tumor necrosis factor–α, interleukin-4, c4 complement, interleukin-6, and interleukin-10.Malaria is caused by the bite of  Plasmodium species, including P. falciparum, P. vivax, P. ovale curtisi, P. ovale wallikeri, P. malariae, and P. knowlesi [Wuchereria bancrofti), onchocerciasis (caused by Onchocerca volvulus), and loiasis (caused by Loa loa) are the most common filarial infections in sub-Saharan Africa [Wuchereria bancrofti, lymphatic filariasis can be caused by Brugia malayi and Brugia timori in a lesser proportion [Mansonella perstans, is the cause of Mansonellosis, and most patients infected with M. perstans have been reported to be asymptomatic, unlike those with loiasis [Malaria is a disease caused by the protozoa genus knowlesi . Accordiknowlesi . Filarian Africa –5. In adoportion . Approxioportion ,8; in adoportion . Another loiasis .Anopheles gambiae, An. arabiensis, An. merus, An. merus, An. funestus in Afroca; An. philippinensis and An. barbirostris in India and Bangladesh; An. leucosphyrus, An. barbirostris, An. donaldi, An. letifer, An. whartoni, An. macualtus, An. campestris in Malaysia; An. minimus in the Philippines; An. balabacensis in Indonesia; An. farauti and An. punctulatus in Papua New Guinea; An. koriensis in the Solomon Islands; An. darlingi and An. aquasalis in South America; and An. sinensis in China and Korea [Malaria and filariasis are significant vector-borne diseases that are co-endemic in the same human populations. The common vectors are nd Korea . Conseqund Korea . To datend Korea . BecauseThe systematic review protocol was registered at PROSPERO (registration No. CRD42022334494). The results of the systematic review and meta-analysis followed the Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) 2020 statement .The systematic review question followed the PICO  question. P included participants who were enrolled in the studies for testing malaria and filariasis parasites by any method, I was not applied, C was not applied, and O was the prevalence of co-infections.We searched five electronic databases  for peer-reviewed articles published between January 1, 1898, and May 22, 2022, in the English language. An additional search was performed in Google Scholar. The search strategy used the following key terms and their appropriate synonyms: (1) malaria, AND (2) filariasis, AND (3) . For searching in PubMed, we used Medical Subject Heading terms to help search with key terms to retrieve potentially relevant studies. We included primary observational studies (prospective or retrospective) and cross-sectional studies to examine the pooled prevalence of malaria and filariasis co-infections among enrolled human participants. In addition, we included cohort and case-control studies to identify the difference in characteristics between co-infections and malaria/filariasis monoinfection. Malaria is diagnosed by microscopy, rapid diagnostic test (RDT), or molecular methods. Filariasis is diagnosed by direct microscopy, leukoconcentration, circulating filarial antigen (RDT), or molecular methods. A patient was considered to have concomitant infections of malaria and filariasis if positive for both malaria and filaria parasites by the above tests. We excluded studies without full-text , review articles ; case reports; mosquito studies; studies reporting malaria intervention measures, vector co-infections, transmission dynamics, or the development of techniques for malaria/filariasis identifications; studies from which we were unable to extract the data of co-infections; studies with spatial statistics demonstrating co-infections; in vivo or in vivo studies; studies conducted by the same authors with overlapping participants; and conference abstracts.Two review authors  independently performed the study selections. All titles and abstracts, followed by the full texts of potentially relevant studies, were screened, and relevant studies were included for full-text screening against eligibility criteria. Discrepancies between the two review authors during the study selection process were resolved by discussion to reach a consensus. Two reviewer authors  extracted the following data into a Microsoft Excel spreadsheet : names of the first authors, publication year, study sites, time for conducting the study, study design, number and characteristics of participants, number and characteristics of patients with co-infections, malaria and filariasis parasites, characteristics of co-infections, and diagnostic method for malaria and filariasis parasites.Two review authors  assessed the quality of all included studies using the critical appraisal tools of the Joanna Briggs Institute (JBI) . The JBII2), (4) the weight of each study, and (5) the number of cases for meta-analysis of the pooled prevalence. To identify the possible source(s) of heterogeneity of the pooled prevalence, we conducted subgroup analyses using study area, study design, participants, febrile conditions, age groups, diagnostic methods for malaria, and diagnostic methods for filariasis. Publication bias was assessed by visualizing the funnel plot asymmetry, Egger’s test, and the contour-enhanced funnel plot. STATA 14.2 was used for all statistical analyses .We estimated the pooled prevalence and probability of malaria and filariasis co-infections among participants included in the studies using random-effects meta-analyses by DerSimonian and Laird . The remA total of 951 articles were identified through database searching: 331 from Embase, 268 from PubMed, 182 from Scopus, 135 from Medline, and 35 from CENTRAL. After 352 duplicates were removed, 599 articles were screened for titles and abstracts. Then, the remaining 465 articles were excluded due to nonrelevant studies. The remaining 136 articles from five databases and eight relevant articles identified from Google Scholar were assessed for eligibility. A total of 118 full-text articles were excluded, with reasons: 15 without full texts, 13 review articles, 12 with no co-infected cases, 11 with only filariasis infections, 10 mosquito studies, 8 with only malaria infection, 6 antibody responses to malaria/filariasis infection, 6 case reports, 6 malaria intervention measures, 5 co-infections in vectors, 4 transmission dynamics, 4 developing techniques for malaria/filariasis identifications, 4 from which we were unable to extract the data of co-infections, 4 spatial statistics demonstrating co-infections, 3 with no malaria or filariasis case, 2 in vivo studies, 2 studies conducted by the same authors with overlapping participants, 1 knowledge/attitudes/perceptions, 1 in vitro study, and 1 conference abstract. The 24 articles that met the eligibility criteria were included in the systematic review .One cohort study was of high quality , and anoI2, 96.7%, 17 studies; We estimated the pooled prevalence of malaria and filariasis co-infections among participants using the data available from 17 studies –38. ResuI2: 96.8%, 12 studies), 0.2% in Asia , and 1.6% in South America . Subgroup analysis of the study designs demonstrated that the prevalence of co-infections among participants enrolled in cross-sectional, prospective observational, and retrospective observational studies was 1.5% , 0.1% , and 2.3% , respectively. Subgroup analysis of participants demonstrated that the prevalence of co-infections among participants in communities and participants in hospitals/clinics was 1.6%  and 1.0% , respectively. Subgroup analysis of febrile conditions demonstrated that the prevalence of co-infections among febrile, both febrile and afebrile, and not specified for febrile conditions was 0.2% , 1.0% , and 2.6% , respectively. Subgroup analysis of age groups demonstrated that the prevalence of co-infections among studies enrolling participants of all age groups and only children was 0.8%  and 5.5% , respectively. Subgroup analysis of diagnostic methods used for malaria demonstrated that the prevalence of co-infections among studies that used the microscopic method, molecular method, microscopy/RDT, RDT/molecular method, and microscopy/molecular method was 1.2% , 5.1% , 0.1% , 0.8% , and 2.9% , respectively. Subgroup analysis of diagnostic methods for filariasis parasites demonstrated that the prevalence of co-infections among studies that used microscopic method, molecular method, direct microscopy/leukoconcentration technique, RDT, and microscopy/RDT was 0.3% , 4.0% , 2.9% , 0.9% , and 3.6% , respectively.W. bancrofti co-infections among participants was estimated using the data available from 11 studies [W. bancrofti co-infections among participants was 0.7% , 0.2% , and 1.6% , respectively. Subgroup analysis of study designs demonstrated that the prevalence of malaria and W. bancrofti co-infections among participants enrolled in cross-sectional and prospective observational studies was 1.0%  and 0.1% , respectively. Subgroup analysis of study designs demonstrated that the prevalence of malaria and W. bancrofti co-infections among studies that enrolled participants in communities and hospitals/clinics were 1.0%  and 0.1% , respectively. Subgroup analysis of febrile conditions demonstrated that the prevalence of malaria and W. bancrofti co-infections among studies that enrolled specified and nonspecified febrile conditions were not specified was 0.2%  and 1.5% , respectively. Subgroup analysis of age groups demonstrated that the prevalence of malaria and W. bancrofti co-infections among studies with participants of all age groups and those with children only was 0.3%  and 1.2% , respectively. Subgroup analysis of diagnostic methods for malaria demonstrated that the prevalence of malaria and W. bancrofti co-infections among studies that used the microscopic, microscopy/RDT, and RDT/molecular methods was 1.2% , 0.1% , and 0.6% , respectively. Subgroup analysis of the diagnostic methods for filariasis parasites demonstrated that the prevalence of co-infections among studies that used the microscopic, RDT, and microscopy/RDT methods was 0.2% , 0.9% , and 3.6% , respectively.Results of subgroup analyses are shown in L. loa co-infections among participants was estimated using the data available from five studies [L. loa co-infections was 1.2%  depended on continents, study design, participants, febrile conditions, age groups, diagnostic methods for malaria, and diagnostic methods for filariasis  and comparable levels of CX3CL1, CXCL7, CXCL9, CXCL11, and CCL28 compared with malaria monoinfection. Olaniyan et al. [Eight studies reported the characteristics of co-infections of malaria and filariasis co-infections ,33,39,40n et al.  conducten et al.  and founn et al. .Four of the eight studies (50%) reported differences in clinical and laboratory characteristics between patients with co-infections and those with malaria monoinfection. Dolo et al.  performeP < 0.455). The contour-enhanced funnel plot demonstrated that the effect estimates were in both significant and nonsignificant areas , L. loa (39%), and P. falciparum (33%) co-infection was reported in Cameroon. Another study in Tanzania that demonstrated the highest rate of co-infections (14%) suggested that the co-infections were caused by the availability of the mosquito vectors in the area, including An. gambiae and An. funestus. Therefore, the prevalence of malaria and filariasis co-infections might be high according to the high prevalence of both diseases with the abundance of the vectors in the same area.In this study, we found that the participant-pooled prevalence of malaria and filariasis co-infections was very low at 0.7%. Nevertheless, the prevalence of this co-infection varied with several factors, such as geographical location, study design, participants, febrile condition, age group, diagnostic method for malaria or filariasis, and different filarial parasites. Therefore, it is postulated that the rate of malaria and filariasis co-infections may be higher in areas where both diseases are highly endemic. This hypothesis was supported by the subgroup analysis, which demonstrated a higher prevalence of co-infections in Africa (2.2%) than in Asia (0.2%). The highest prevalence of co-infections by country included Cameroon (12.7%) , India . However, a higher prevalence of co-infections was reported with M. perstans (1.0%) and L. loa co-infections (1.2%). The low prevalence of malaria and W. bancrofti co-infections could be attributed to the difficulty in detecting W. bancrofti in blood smear because of its nocturnal subperiodic nature of circulation, particularly in low parasitemia [M. perstans was often asymptomatic and was detected in only febrile patients; meanwhile, L. loa was present more often in both febrile and afebrile individuals [The meta-analysis of the prevalence of co-infection by filarial parasites showed that the pooled prevalence of malaria and asitemia . In addiasitemia ; therefoividuals .Trichuris mostly affected patients aged 8 to 16 years [The distribution of concomitant infections may be attributable to differences in behavior and occupation between age groups. In their study, Bisanzio et al. found that filariasis and hookworms mostly affected adults, but malaria, schistosomiasis, and 16 years . This fi16 years . Adults 16 years .Anopheles species can transmit both malaria and filariasis [An. gambiae, An. arabiensis, An. merus, An. merus, and An. funestus are the major Anopheles species that transmit malaria and filariasis. However, An. philippinensis and An. barbirostris are the main vectors for malaria and filariasis in India and Bangladesh [An. gambiae infected with Wuchereria than in noninfected mosquitoes, indicating that W. bancrofti infection may increase mosquito susceptibility to P. falciparum infection [Anopheles species had higher P. falciparum infection rates than those of W. bancrofti and thus described the more extended latent period of W. bancrofti in the vector [Co-infections in malaria and other parasites were reported previously and occurred by chance in varied geographic regions . The prelariasis , but thengladesh . There wngladesh ,43,44. Tngladesh . Anotherngladesh . Therefongladesh ,46. In angladesh . Muturi nfection . Anothere vector .P. falciparum parasite was lower in those with co-infection than in those without filariasis [As for our qualitative synthesis, distinct clinical characteristics of patients with co-infections compared with those with malaria monoinfection were at increased risk of having an enlarged spleen, lower rate of severe anemia, and more likely to be asymptomatic. Moutongo Mouandza et al. suggested that co-infections may protect patients from anemia by reducing the contribution of the inflammatory immune response . Distinclariasis , and Mutlariasis . Finallylariasis . HoweverThe present study had some limitations. First, the number of included studies was low; however, the results of pooling data from these studies would help provide the current status of the diseases and may indicate the interference of malaria on eliminating filariasis. Second, the characteristics of malaria and filariasis co-infections were also limited because of the limited investigations of these parasites’ co-infection in the literature. Limitations on the details of the cytokine levels in co-infections were based on one-off studies. Even though these are important observations, it may not be prudent to generalize the cytokine expression levels in co-infections based on these studies. In addition, the population size was greatly reduced and insufficient for generalization, for example, for a continent. Third, there was heterogeneity in the pooled prevalence and probability of co-infections among participants. Therefore, careful interpretation of the pooled prevalence of co-infections is recommended. Fourth, because publication bias among the studies included in the meta-analysis of co-infection was less likely, we did not perform the trim-and-fill method to correct the pooled effect estimate.The present study showed that the prevalence of malaria and filariasis co-infections was low and varied between geographical areas in the selected articles. Co-infections tended to occur with a low probability. Because few details on the characteristics of malaria and filariasis co-infections have been reported in the literature, further studies investigating the outcomes and characteristics of co-infections are needed if co-infections become a health problem.S1 PRISMA Abstract Checklist(DOCX)Click here for additional data file.S2 PRISMA 2020 Checklist(DOCX)Click here for additional data file.S1 Table(DOCX)Click here for additional data file.S2 Table(XLSX)Click here for additional data file.S3 Table(DOCX)Click here for additional data file.


The WHO recommends continuous surveillance of malaria in endemic countries to identify areas and populations most in need for targeted interventions. The aim of this study was to assess the prevalence of malaria and its associated factors among first antenatal care (ANC) attendees in rural Burkina Faso.A cross-sectional survey was conducted between August 2019 and September 2020 at the Yako health district and included 1067 first ANC attendees. Sociodemographic, gyneco-obstetric, and medical characteristics were collected. Malaria was diagnosed by standard microscopy and hemoglobin level was measured by spectrophotometry. A multivariate logistic regression analysis was used to identify factors associated with malaria infection.Overall malaria infection prevalence was 16.1% (167/1039). Among malaria-positive women, the geometric mean parasite density was 1204 [95% confidence interval (CI) 934–1552] parasites/µL and the proportion of very low (1–199 parasites/µL), low (200–999 parasites/µL), medium (1000–9999 parasites/µL) and high  parasite densities were 15.0%, 35.3%, 38.3% and 11.4%, respectively. Age < 20 years (adjusted odds ratio (aOR): 2.2; 95% CI 1.4–3.5), anemia (hemoglobin < 11 g/deciliter) , the non-use of bed net , and the absence of intermittent preventive treatment with sulfadoxine–pyrimethamine  were positively associated with malaria infection.P. falciparum malaria infection at their first ANC visit. Strengthening malaria prevention strategies during the first ANC visit is needed to prevent unfavorable birth outcomes.The study showed that one out of six pregnant women had a microscopy-detected  Plasmodium parasites, can lead to severe pregnancy outcomes such as maternal and fetal anemia, stillbirth, intrauterine growth retardation and preterm births, with the latter two being major contributors to low birth weight (LBW), a strong precursor for neonatal mortality ) compared to those who used an ITN (14.5% [95% CI 12.2–17.1%]), with however a non-significant difference (p = 0.19). Women without any dose of SP before the visit had a higher proportion of malaria infections (17.2% [95% CI 14.9–19.8%]) compared to those who had a dose of SP . Plasmodium falciparum was the only species detected during the thin blood smear examination. The geometric mean of parasite density was 1204 [95% CI 934–1552] parasites/µL. The proportion of very low (1–199 parasites/µL), low (200–999 parasites/µL), medium (1000–9999 parasites/µL) and high  parasite densities were 15.0%, 35.3%, 38.3% and 11.4%, respectively. The mean hemoglobin level was 10.5 (± 1.4) g/dL, and nearly 60.0% of women were anemic (hemoglobin level < 11.0 g/dL) at their first ANC visit.Overall, 16.1% (167/1039) (95% CI 13.4–18.5%) of participants had a microscopy-detected malaria infection. The prevalence of malaria was significantly higher among women aged below 20 years  compared to those ≥ 20 years  (p < 0.05)  Table . After a5) Table .Table 3BP. falciparum infection of 15.7% (95% CI 12.3–20.2%) during the second and third trimesters of pregnancy [Nearly one out of six pregnant women attending their first antenatal clinic visit for their ongoing pregnancy had a microscopy-detected malaria infection in our study setting. This suggests that prevention strategies should be strengthened as malaria infection in early pregnancy is associated with a higher risk of unfavorable birth outcomes , 17. Theregnancy . Thus, iregnancy , 25.4% iregnancy , 59.7% iregnancy –24. Thisregnancy . Thus, iThe geometric mean parasite density was much higher than that reported in Ghana (442 parasites/µL) and Cameroon (529 parasites/µL) . HoweverP. falciparum was the only species reported in this study. Such a result was not surprising and corroborates previous findings that reported P falciparum as the main species involved in malaria in the country [P. falciparum is the deadliest malaria parasite and the most prevalent on the African continent [P. falciparum infection prevention measures is needed to control malaria’s impact on pregnancy outcomes. country . This coontinent . Thus, aThe proportion of women that used an ITN the night prior to their visit to the antenatal clinic was surprisingly high (80%), given that ITN distribution to pregnant women usually occurs at the first ANC visit. This may be because almost 70% of the pregnant women were secundigravid or more, suggesting they may have already been exposed to ANC service and received ITN. The prevalence of malaria infection was significantly higher among women who did not use an ITN the night before they visited the antenatal clinic. This finding is in line with previous reports demonstrating the effectiveness of ITN in preventing malaria in pregnancy . Pre-conAlthough SP use is not indicated during the first trimester of pregnancy, nearly one out of ten pregnant women had their first dose prior to their first ANC visit, and the risk of malaria infection was lower among women who had SP before the first ANC. Given that the mean gestational age at first ANC was nearly 22 weeks in our study, strategy is thus needed to cover pregnant women as early as 12 to 16 weeks of gestation to benefit from the full potential of the IPTp-SP strategy . In addiIn this study, adolescent pregnant women were found to have a higher prevalence of malaria infection with up to a twofold increase. This is in line with studies conducted in different sub-Saharan African countries which reported that young pregnant women are at the greatest risk of malaria infection and have the highest parasite densities , 38, 39.In this cross-sectional survey, we described the prevalence of malaria infection during the first ANC contact in a rural health district of Burkina Faso. However, some limitations are worth noting. Indeed, pregnant women were not followed up to evaluate the effect of malaria infection during the first ANC visit on birth outcomes. In addition, gestational age was assessed using the pregnant women's knowledge of their last menstrual period or the fundal height measurement in most cases due to the absence of ultrasounds, and this is frequently prone to many errors. Furthermore, the standard microscopy diagnosis approach used for this study may have underestimated the actual prevalence, considering that submicroscopic malaria infections are common in endemic areas. However, the study has highlighted the burden of malaria infection at the first ANC contact among pregnant women and suggested strategies to mitigate malaria's impact on pregnancy outcomes.The study showed that one out of six pregnant women had malaria infection at their first antenatal care visit. This rate was even higher among pregnant adolescents, with one in three cases of malaria infection at their first ANC. Adequately preventing infection during that period of the pregnancy could substantially benefit pregnant women and their unborn fetuses.