CD209 and CD28 genes act as co-stimulators and regulators of the immune system, while the STAT6 gene has been reported to mediate cytokine-induced responses. Single nucleotide polymorphisms of these genes might lead to differential disease susceptibility among populations at risk for malaria, due to alterations in the immune response. We aim to identify key drivers of the immune response to malaria infection among the three SNPs: CD209 (rs4804803), CD28 (rs35593994) and STAT6 (rs3024974). After approval and informed consent, we genotyped blood samples from a total of 531 children recruited from Nigeria using the Taqman SNP genotyping assay and performed comparative analysis of clinical covariates among malaria-infected children. Our results reveal the CD209 (rs4804803) polymorphism as a susceptibility factor for malaria infection, significantly increasing the risk of disease among children, but not CD28 (rs35593994) or STAT6 (rs3024974) polymorphisms. Specifically, individuals with the homozygous mutant allele (rs4804803G/G) for the CD209 gene have a significantly greater susceptibility to malaria, and presented with higher mean parasitemia. This observation may be due to a defective antigen presentation and priming, leading to an ineffective downstream adaptive immune response needed to combat infection, as well as the resultant higher parasitemia and disease manifestation. We conclude that the CD209 gene is a critical driver of the immune response during malaria infection, and can serve as a predictor of disease susceptibility or a biomarker for disease diagnosis.Malaria remains a significant disease, causing epic health problems and challenges all over the world, especially in sub-Saharan Africa.  Plasmodium falciparum, with multiple studies implicating the asexual blood stages of the parasite for its infection pathology [P. falciparum, in context of the host’s genetic contribution to disease outcome, is a complex process, and requires stepwise dissection.Malaria remains a parasitic disease with about 3.2 billion people at risk of infection ,2, with athology ,20. The CD209 gene, also known as the dendritic cell-specific intercellular adhesion molecule-3-grabbing non-integrin (DC-SIGN) gene, encodes a transmembrane receptor on dendritic cells, and is a significant player in recognizing and presenting pathogens with a diverse evolutionary origin [rs4804803 (snp -336G/A) gene promoter polymorphism, have been shown to modulate disease susceptibility or severity [To establish a protective immune mechanism against infection, there is the need for an intricately organized, innate and adaptive response, involving dendritic cells, macrophages, and B and T lymphocytes ,21,22. Ty origin ,24. The y origin , as wellseverity ,30,31,32severity . ImportaCD28, on the other hand, is an important costimulatory molecule, promoting transcription signaling, activation and differentiation of naive CD4 and CD8 T cells, as well as activation of cytokines or cytokine receptors [CD28 in protective immune responses against various diseases [eceptors ,34. Studdiseases ,36,37,38diseases . TherefoSTAT6), enhancing IL-12 production in dendritic cells and driving a Th1 immune response in STAT6-/- mice [STAT6 in the immune response to malaria infection is currently limited to a handful of studies, with no definitive conclusion [STAT6 and FOXP3 among malaria-infected children [STAT6 promoter variant rs3024944 being associated with uncomplicated malaria, and the FOXP3 SNP rs11091253 being associated with parasitemia. The role of these regulatory variants in association with disease or covariates of infection has been very challenging to elucidate.Furthermore, published reports have shown that IL-10 production in response to a pathogenic stimulus is modulated by signal transducer and activator of transcription 6  [CD14 gene polymorphisms are only one component of the complex host–parasite interaction, and thus constitute only a piece of the genetic puzzle required for a complete picture of the immune response to malaria. Considering the paucity of available data, what are the roles of CD209, CD28 and STAT6 gene polymorphisms, either individually or in combination, with malaria infection among children, and how are they associated with markers of disease severity ? Do these polymorphisms serve as susceptibility factors for malaria infection, or do they play any critical role in regulating disease covariates such as anemia and parasitemia in West Africa? To answer these questions, we evaluated the genetic variability of CD209 (rs4804803), CD28 (rs35593994) and STAT6 (rs3024974) single nucleotide polymorphisms among malaria-infected children in Nigeria and extrapolated any association with markers of infection. Our data suggest that CD209 (rs4804803) is a susceptibility factor for clinical malaria that also influences disease pathogenesis, but not CD28 (rs35593994) or STAT6 (rs3024974). We also show that despite the preponderance of CD28 (rs35593994) wild type variants that would assist in T cell differentiation and parasite clearance, this was not the case. We postulate that a defective antigen presentation, depicted by the higher frequency of CD209 mutant alleles, renders downstream immune response and parasite clearance moot. It also appears that any role for STAT6 (rs3024974) variants to influence malaria pathogenesis occurs via a completely different mechanism, and this polymorphism is potentially still in flux.We have previously demonstrated the importance of the 9190C/T)  in malarThis study population consisted of febrile children recruited at St Mary Catholic Hospital, Ibadan, and in a rural primary health care center , southwest Nigeria, from November 2013 to November 2014, and in accordance with the 1975 Helsinki declaration. Detailed demographic, geographic and clinical parameters, as well as the genomic DNA extraction process, including modifications, are as described in . Approval was obtained from the University of Ibadan/University College Hospital Institutional Review Committee . Two hundred and thirty-one children presenting with malaria infection, for whom informed consent was obtained, were included into the study. Additionally, 330 matched uninfected children were recruited as controls, as part of the original study. Genomic DNA quality was accessed with a Nanodrop 2000 . Axillary body temperature (in Celsius) and packed cell volume (expressed as a percentage), indicative of fever and anemia, respectively, were determined. Parasite count was evaluated per international standards .STAT6 , alongside pre-made assays for CD209  and CD28  SNPs, were utilized. Briefly, a 10 µL reaction mixture was prepared for each assay containing 1 µL of the 20X Taqman SNP genotyping assay, 5 µL of 2× Taqman Mastermix , 1 µL of 20 ng genomic DNA and 3 µL of nuclease-free water. The reactions were set up for SNP genotyping using the CFXconnect  real-time PCR machine with the following reaction conditions: 90 °C for 10 min, followed by 90 °C for 30 s, 56 °C for 30 s and 72 °C for 50 s, with a final extension of 72 °C for 5 min. Melt analysis was performed to confirm assay specificity; temperatures varied from 65 °C to 95 °C with increments of 0.5 °C for 5 s. We generated allele calls with the CFX Manager software.To perform the SNP genotyping analysis, our designed custom assay for CD28 (rs3559399), CD209 (rs4804803) and STAT6 (rs3024974), with SNP’s rejected on the threshold of p < 0.05, as described by [The program SNPstats  was usedribed by ; associaSTAT6 (rs3024974), CD28 (rs35593994) and CD209 (rs4804803) gene polymorphisms using Taqman SNP genotyping assays. We also performed association analyses of genetic variants with clinical variables among the malaria-infected group. The genotypic frequencies of STAT6, CD28 and CD209 gene promoter polymorphisms in malaria patients were compared to the control group with logistic regression analysis  and CD209  but not STAT6  gene polymorphisms. Although there was no significant difference in genotypic frequency between groups for the STAT6 (rs3024974) gene, we did however observe a higher frequency of wild type G/G (74.4%) and heterozygote G/A (20.1%) variants in the control group, while the opposite (higher frequency of homozygous recessive variant A/A) was seen in the malaria group  for analysis . Our resectively .CD28  gene polymorphism, we observed a higher frequency of homozygous mutant variants (-372A/A), though statistically insignificant in the control group compared to the malaria-infected group  (-372G/A) were present in the malaria group (65.5%) compared to the control group (28.2%). Similarly, significantly more mutant variants (-383G/G) were observed in the malaria group (46.0%) compared to the control group (22.7%) for the CD209  gene, depicting the homozygous mutant variant as a susceptibility factor for clinical malaria among children in southwestern Nigeria. This observation was not seen for other genes, except for CD28 gene heterozygous variants (372G/A). The opposite was the case among those with the CD209 gene homozygous dominant variant (-383A/A)  (Examining genotypic frequency of the ctively) ; howeverctively) .STAT6 (rs3024974) gene polymorphism did not increase the risk of malaria with the dominant  or recessive allele . However, in the case of the CD28 (rs35593994) gene polymorphism, the dominant allele  demonstrated a significant increase in risk of malaria infection (p = 0.0001) but not the recessive allele  (CD209 (rs4804803) gene polymorphism, a significantly increased risk of malaria infection was observed with both dominant and recessive alleles  . In the GGA; H3: GAG; H4: GGG; and H8: AGG) showed a significant difference in haplotype frequencies between groups; others were insignificant. Based on the odds ratio, the same haplotypes  in addition to haplotype H5  showed the highest/increased risk of malaria compared to others (CD28 (rs35593994) and CD209 (rs4804803) gene polymorphisms in the malaria group  gene polymorphisms presented the strongest association with malaria, we set out to examine the possibility that its genetic variants may be modulating the clinical covariates associated with disease , packed cell volume, PCV (indicative of anemia) and parasitemia). Comparing wild type versus homozygous mutant variants, we report significant differences (rs4804803A/A < rs4804803GG) with age . A similar observation with age and parasitemia, in addition to PCV (p = 0.005) was made when we compared patients with wild type variants (rs4804803A/A) to those with heterozygous (rs4804803A/G) variants (STAT6 (rs3024974) gene showed a significant correlation between age and all its variants, and to some degree, with temperature, PCV and parasitemia (STAT6 gene promoter (rs3024974G/A) presented with the most protection against malaria infection , possibly implying that the evolution of this gene in the context of malaria infection is still ongoing.Since the variants . To our asitemia . The hetPlasmodium falciparum, the most virulent of the species, is endemic. Protective immunity can be acquired naturally after consecutive malaria infections when dendritic cells, serving as antigen-presenting cells, prime and activate the T cells necessary for downstream adaptive response, for which CD209 and CD28 molecules are significant participants [STAT6 gene is known to regulate the expression of regulatory T cells [STAT6 (rs3024974), CD28 (rs35593994) and CD209 (rs4804803) gene polymorphisms in modulating disease susceptibility and infection outcome, as well as their relationship to clinical covariates of disease among children diagnosed with malaria in southwestern Nigeria. Remarkably, we show that the CD209 (rs4804803) gene polymorphism is a susceptibility factor for malaria among the three genes under study, showing a significant disease-related association. The majority of malaria-infected children had the homozygous mutant variant (rs4804803G/G), while the control (uninfected) individuals predominantly had the wild type phenotype (rs4804803A/A), confirming our hypothesis that the CD209 gene is a susceptibility factor for clinical malaria.Malaria is a life-threatening disease, causing global morbidity and mortality among endemic populations and non-immune individuals travelling to such locations, with the most deaths among children under five years of age ,4,46. Reicipants ,48,49. L T cells , while p T cells ,42. Ther T cells ,51. To tCD209 encodes dendritic cell-specific ICAM3-grabbing non-integrin (DC-SIGN), and has been shown to bind various ligands including T-lymphocytes, thus contributing to activation of signal transduction pathways and recognition of infectious agents. Several studies have shown its polymorphic variants to be associated with tolerance or susceptibility to diseases, such as tuberculosis, schistosomiasis, leishmaniasis, and sickle-cell disease [CD209 gene has a considerable impact on malaria pathogenesis. Under normal circumstances, this gene protects against infection, but among the significant number of individuals with the mutant variant, this protection is lost, rendering these individuals more susceptible to disease. We show that a defective CD209 response  presented with significantly higher parasitemia and anemia (indicated by a low PCV) compared to the wild type variant, demonstrating its role in driving immune responses to malaria, more so than STAT6 (rs3024974) or CD28 (rs35593994) polymorphisms. This observation complements our previous report showing that the preponderance of the G mutant allele leads to a defective antigen presentation, weakening protective immune responses [ disease ,51,52,53esponses  and, as CD28 gene polymorphism, whereby the heterozygous variant was most common among malaria patients, and the homozygous dominant was most common among controls, this does not appear to be a factor in malaria pathogenesis. This observation implies a balanced immune response, expected to elicit a proper T cell response and control of parasitemia. The fact that this is not the case confirms our suspicion that higher parasitemia among malaria patients is hinged on the preponderance of the CD209 mutant G allele, mediating a poor antigen presentation and an unmitigated disaster in the immune response. For the STAT6 (rs3024974) gene, our analysis did not show any significant diversity in the genotypic frequency of the homozygous dominant (G/G), heterozygote (G/A) and the homozygous recessive (A/A) variants between the malaria and control groups (rs3024974G/G > rs3024974G/A > rs3024974A/A), except when analyzed in the context of disease covariates. The odds ratio showed no indication of an increased risk of malaria infection with the STAT6 (rs3024974) gene polymorphism. This observation, however, contradicts previous studies associating this polymorphism with malaria pathogenesis among Congolese children [STAT6 (rs3024974) polymorphism did not have any effect on disease susceptibility. Rather than driving a severe disease outcome as reported from Ghana, we propose a heterozygous advantage among malaria patients when analyzed with disease covariates. Patients with this variant, despite being the youngest (mean age: 46 months) and therefore expected to show the most severe disease outcome, had the lowest mean parasite count . This observation demonstrates the significantly higher relative fitness in endemic communities, endowed by the heterozygote genotype, but not seen in dominant or recessive groups, similar to that conferred by the sickle cell trait [Though we found a notable diversity in children , as wellchildren . As showll trait ,54.CD28 (rs35593994) homozygote recessive (A/A) variant in the malaria group, though statistically insignificant, implying that this variant possibly has a protective effect against infection. A published report has shown that low level parasitemia is retained in CD28 knockout mice infected with malaria [CD28, and that CD28-deficient mice showed efficient type 1 and type 2 responses during infection with Leishmania major and Heligmosoides polygyrus, respectively [G/A variant in the malaria group might imply a potential additive effect producing a negative interaction of allele G and A, leading to a higher risk of disease in infected but not control groups. Therefore, the heterozygote variant in this case may have a negative effect on the immune response to disease and contribute to susceptibility, while the recessive variant (A/A) seems protective. Teutsch et al. (2004) described the CD28 (rs35593994) SNP through identification of transcription factor binding sites, showing that the A allele possesses a CCAAT enhancer-binding protein site, which is lacking in allele G. Additional reports showed that this variant influences gene transcription and immune alteration in breast cancer [On the other hand, we report a low genotypic frequency of the CD28 rs393994 homectively ,57. The t cancer ,59. We rCD209 (rs4804803) and CD28 (rs35593994) polymorphisms are significantly linked, justifying their co-stimulatory activity with pro-inflammatory cytokines such as IL-4, IL-10, IL17F and TNF-α. In the same vein, the haplotype combinations that identified haplotypes H2 (GGA), H3 (GAG) and H5 (AAA) as important for malaria might be as a result of the additive effect of the three loci under study favoring disease susceptibility. Importantly, the mutant allele G is significantly implicated among the haplotype definitions, implying that a change from an A to G as in the CD209 (rs4804803) gene polymorphism will hamper downstream expression of the gene, thereby affecting the immune response and contributing to malaria pathogenesis, which was not seen in the STAT6 (rs3024974) polymorphism. However, it is not impossible that STAT6 (rs3024974) and CD28 (rs35593994) polymorphisms may mediate malaria pathogenesis through a different mechanism not examined in this study. Overall, the CD209 (rs4804803) gene polymorphism presenting the mutant allele G is critical in determining disease susceptibility, thereby identifying this gene and its polymorphism  as an important driver of immune response during infection.Interestingly, our linkage disequilibrium analysis revealed that


Malaria is one of the major public health problems in many tropical developing countries including Cameroon. Impregnated mosquito bed nets are one of the control measures put in place by the WHO and adopted by the Cameroon's Ministry of Public Health to fight against malaria in pregnancy. This study was a population-based cross-sectional study that investigated the level of adherence, respondent's knowledge, altitude, and practices toward malaria prevention and control.  To investigate this, a sample size of 410 pregnant women who were inhabitants of Foumban Subdivision was examined. Data on net ownership versus usage, pregnancy status, and socioeconomic background were collected using a questionnaire. Parasitological tests for malaria parasites were carried out using peripheral blood samples obtained from finger pricks of the pregnant women for the preparation of thick blood smear and RDTs.  Two hundred and eighteen tested positive (53.4%) with the highest prevalence occurring during the first trimester (79.6%) and in primigravidae (68.8%). Participants believed that mosquito bed nets can protect them against malaria infection. The highest number (81.0%) of the women who had mosquito nets acquired them during antenatal visits. Among those who possessed nets, 42.7% adhered to sleeping under them and few (50%) experienced problems of sweating, discomfort, and heat. Also, the study revealed a high prevalence rate of 63.8% for those who did not use nets during pregnancy as compared to those who owned and used them.  The findings indicated that increased access to impregnated mosquito bed nets is required to lower the risk of malaria infection amongst pregnant women. The Cameroon government should improve health education to families within the locality and pursue an integrated approach to fight against mosquitoes during the rainy season. It has been evaluated and estimated that by the end of the 21st century, 48% of the global population will still be living and exposed to the risk of malaria infection [Malaria is a life-threatening chronic parasitic disease for nonimmune individuals and is spread by the bite of a female Anopheles mosquito, which results in the infection of red blood cells . The vecnfection .Plasmodium falciparum [In Cameroon, over 90% of Cameroon's population are at risk of infection by malaria  with abolciparum .This disease can affect everyone, but individuals at high risk are children under five, pregnant women, people living under emergency situations, as well as immunosuppressed individuals. It has the main consequence in pregnancy being anaemia. Apart from anaemia, malaria infection can also lead to the accumulation of parasites in the placenta, foetal exposure, congenital infection, infant anaemia, infant morbidity and mortality, as well as maternal death . It is dGiven that Foumban Subdivision falls in the area of stable transmission in Cameroon where malaria infection is asymptomatic and also the majority of these pregnant women when pregnant start antenatal consultation late or not at all and this predisposes them to malaria infection. Taking into consideration the methods of control put in place by the WHO, this study investigated the impact of impregnated mosquito bed nets on the prevalence of malaria among pregnant women in the Foumban Subdivision, West Region of Cameroon.The study was conducted in Foumban Subdivision; headquarters of Noun Division which is the largest Division in the West Region of Cameroon. It is a city found in the North East of Bafoussam. Foumban Subdivision is located 5°43′-5°43′ north latitude and 10°53′-10°55′ east longitude and is spread on 7000 ha on 900-1200 m of altitude on average.This was a population-based cross-sectional study using the convenience sampling method in which pregnant women in their households were recruited until a total number of sample size was achieved. During the course of this study, pregnant women who were living in Foumban Subdivision were recruited as participants. A sample size of 410 was calculated based on a 2-sided hypothesis tests using Epi-info with 80% power, confidence interval of 95%, expected frequency for ITNs coverage of 50%, and a precision of 10%, and has added additional 10% to account for nonresponse.Pregnant women who signed a free informed consent and are permanent members of the city were included in the study. Also, only pregnant women with good health status of different age groups were included in the study.For this study, pregnant women who were mentally handicapped or taking antimalarial therapy or who had been treated with antimalaria medications or drugs during the period of study or refused to sign the informed consent were excluded. In addition, it was also excluded those who were not resident in the studied locality or study area.Four hundred and ten participants were randomly recruited in their households from April to July 2018 to participate in this study. Pregnant women of all trimesters, parity, and age group with a mean age of 26.52 ± 7.17 were involved in the study. The majority of the participants were Muslims and the Bamouns were in the majority. Three educational levels were represented, i.e., Primary, Secondary, and Diploma.Pregnant women in their first, second and subsequent pregnancies, different age groups, and different trimesters were recruited in the study after they signed an informed consent form. Data were collected using a standardised questionnaire, RDTs, and prepared thick blood samples for plasmodium detection. A coded number was assigned to participants that were recorded on the questionnaires, the prepared slides, and RDTs.falciparum (HRP2) Card Test was used according to the rules of Clinical and Laboratory Standard Institute for the identification of malaria parasites. Regarding the preparation of thick blood smear, a large drop of blood was placed on a microscopic slide and using the edge of another clean slide spread the blood in a circular manner with 6-15 movements to make an area of 1 cm2. The prepared slides were then air-dried in a horizontal position and placed in the slide box and carefully transported to Foumban District Hospital for parasitological examination. Once in the laboratory, the slides were stained using a 10% Giemsa solution and observed under a light microscope at objective ×100 after adding oil immersion for the identification of malaria parasites.Blood samples were collected from a finger prick after cleaning it with cotton wool soaked in alcohol by the principal investigator. This blood was then used to perform the Rapid Diagnostic Test (RDTs) and thick blood smear. Concerning RDTs, the First Response® Malaria Ag. P. The data obtained was entered and stored into the computer using MS excel software 2007 and was analyzed using SPSS version 20 software. For categorical or qualitative variables, data were presented as frequencies, while continuous variable (age) data were presented as means. A chi-square test was used for comparisons between respondent characteristics and malaria prevalence at 0.05 significant levels. A univariate analysis was also carried out to independently determine whether any of the prospectively defined independent factors  was significantly associated with malaria prevalence. To assess relative risk, odds ratio at 95% confidence interval was computed using Medcalc software.Among the 410 pregnant women who participated in the study, the majority of them were urban residents, Bamouns, and Muslims. About 84.4% of the respondents were married, and most of them were housewives during data collection. Their ages ranged between 14 and 47 years with a mean age of 26.52 ± 7.17. Pregnant women aged between 14-24 years were more represented with 44.9% compared to others. Furthermore, most of the respondents, 180 (43.9%) were housewives, 144 (35.1%) were holding a small business, 32 (7.8%) were student, 26 (6.3%) had no job, 24 (5.9%) were civil servant, and 4 (1.0%) were farmers. About 266 (69.8%) had a secondary level of education, 104 (25.4%) primary education, 3.4% (14) had a higher level of education or at least a diploma, and 6 (1.5%) had no formal education. Regarding the ethnic group of the study population, 344 (88.8%) were Bamoun, 22 (5.4%) Bamelike, 12 (2.9%) Haoussa, 10 (2.4%) were from other tribes, and 2 (0.5%) Borroro.A total number of 202 respondents who did not have mosquito bed net had good knowledge of malaria transmission. Of this total number examined, 67.3% (144) knew that malaria is transmitted through mosquito bites. Respondents also declared that malaria is transmitted through water 18.7% (40) and mangoes 5.6% (12). Others mentioned that they do not know the cause of malaria 8.4% (18). The main reasons of not having mosquito bed nets were financial problems and availability during the period of free mass distribution. At least nine over ten (89.6%) of the respondents declared that if they are given mosquito bed nets they shall use it effectively. Few but not negligible proportion of respondents (10.4%) declared that they could not use ITNs if given to them despite all declared that it is to protect against malaria. Regarding other methods of prevention mentioned by the respondents, use the IRS of insecticide, destruction of breeding sites, the use of insecticide bomb accounted for 1.9% (4) each. Only two respondents declared clearing bushes to fight against transmission while more than 95% were using nothing to prevent malaria as shown in Of 410 respondents, 50.7% (208) did not have mosquito bed net and 49.3% (202) had a mosquito net. The study recorded 81.0% (162) of the respondents who had acquired their net during the antenatal visit, 16.5% (33) from Ministry of public health during free distribution, 2.0% (4) purchased theirs from the supermarket, and 0.5% (1) had from gift. Looking at the net usage, 42.7% (70) used their net and 57.3% (94) did not use theirs during pregnancy. The study also measured the frequency of net usage per week and revealed that 13.4% (18) used one to two times per week, 16.4% (22) used 3 to 5 times a week, 68.7% (92) used more than 5 times a week, and 1.4% (2) had never used theirs. Moreover, we equally examined net usage during the year, and 54.7% (82) said they used their net throughout the year, 44.0% (66) said they used mostly during the rainy season, and 1.3% (2) said is during the dry season. Among these respondents that possessed nets, 58.0% (94) tucked into the mosquito bed net regularly; however, 25.0% (42) tucked in once a while 16.0% (26) had never tuck in. This result is highlighted in Out of 202 participants that had mosquito bed nets, 45% loved using bed nets which have a regular shape, 30% had no choice, and about 25% preferred conically shaped of net as seen in A large portion of respondents, 48% had no problem or challenge during the use of a mosquito bed net. However, 38% of them mentioned that they feel hot when sleeping under mosquito bed nets, while 13% experienced had discomfort and 2% sweated during net usage. This best demonstrated in The respondents who owned mosquito nets indicated several reasons why they were sleeping under them, among which 140 (88.61%) claimed to sleep under the mosquito net in order to protect themselves, 16 (10.13%) were protecting themselves and children, 2 (1.3%) were protecting their children, and 2 (1.3%) were sleeping under the net to avoid malaria. This is well presented in Over 83% of those who were having mosquito bed nets relied on it to fight against malaria. However, 10% used insecticide as another control measure and 7% burnt organic materials. This is presented in Among the 410 pregnant women examined, a total of 218 (53.4%) were found to be positive for malaria parasites. The overall prevalence of malaria among pregnant women was 53.4% as presented in P < 0.005). However, the risk was high for those who did not use the net during pregnancy  had two times the risk of being infected with malaria as compared to those who go to bed early (before 10 pm). There was a significant difference between the two studied parameters.P > 0.05) and both those who wake up early and those wake up late were at the same risk of contracting malaria. This is well demonstrated in These findings also revealed that 54% for those who wake up early (before 6 am) and the trend observed for those who wake up late was slightly lower (51.4%). Also, no significant difference was recorded between the studied parameters (P = 0.05).In this study, 100% of those who had no formal education were infected, 53.8% of those with primary education, 51.4% of those with secondary education, and 71.4% of those who had acquired postsecondary and post-high school education had positive parasitemia . The relP value < 0.05). The risk factor was high for pregnant women belonging to 14-24 and 25-34 years as seen in One hundred and eighty-two respondents aged between 14-24 years were examined; 106 (58.2%) were positive for malaria and had the highest prevalence. There was a significant difference between individuals having 34 years and above and other classes of age (The highest infection rate of 68.8% was recorded for primigravidae followed by secundigravidae at 52.9% while the lowest (47.5%) was recorded in multigravidae . The priA considerable percentage of the respondents were aged between 14-24 years, and majority of them were married (84.4%). About 44% of pregnant were housewives (43.9%), Bamoun 88.8%, and Muslims (87.3%). Those with secondary level education constituted the highest percentage (69.8%).Approximately all the respondents who had mosquito bed nets had good knowledge of the cause and symptoms of malaria 202 (49.3%). Among those pregnant women who did not have net, 144 (67.3%) knew that it was transmitted by mosquito bites and some associated malaria to contaminated water, mangoes, and others do not know the main cause of malaria. This finding corroborates with that of Tassew et al. who demonstrated that the majority of the respondents who participated in his study knew that malaria is transmitted through mosquitoes even though the least number did not know what causes malaria .The study examined bed nets ownership by pregnant women in Foumban Subdivision. Results revealed that over 202 (49.3%) of the participants owned net during pregnancy. This finding corroborated  who repoFurthermore, 93.3% relied on MBNs to fight against malaria and a few number mentioned other preventive measures such as IRS, insecticide bomb, destruction of breeding sites with 1.9% each, and clearing bushes with 1%. This finding is similar to Tassew et al. in Ethiopia who reported that 87% of the household depend on MBNs to fight against malaria. Also, those who were not having MBNs mentioned that if they give they will use it effectively and they also indicated that they relied on MBNs to fight against malaria. More than 67.3% (144) of the studied participants who did not have mosquito bed nets mentioned mosquito bites as the main cause of malaria . This isRespondents who did not have mosquito bed nets were in agreement that nets can protect them against malaria infection. This result is in agreement with the findings of Njoroge et al. who reported that net protects against malaria infection . They alThe study highlighted that pregnant women with low income levels were highly infected with malaria (66.7%) as compared to those with high and average income levels. This finding agrees with , 25 who The highest parasitaemia was reported in respondents who went to bed late (after 10 pm). The time of going to sleep may determine the likelihood of being bitten by mosquitoes, hence infection by malaria. People who stay awake for a long time probably expose themselves to mosquito bites, whereas those who sleep early cover themselves hence reducing the likelihood of the chance of being bitten. This is similar to what was obtained in a study carried out in Kenya by Mukanda . They reThis study also revealed that the highest parasitemia was observed among respondents who wake up early from the bed as compared to those who wake up late. Those who wake early had the highest risk of being infected. These findings have not been confirmed in other studies and need further investigation, though Kline  suggesteWomen who owned mosquito bed nets and used them had less malaria infection as compared to those who had nets but did not use them; hence, there was a significant difference in malaria parasitemia between those who did not use and those who used net during pregnancy. Also, those who had nets but did not use them were at higher risk of being infected. Bed net usage during pregnancy therefore reduces cases of malaria. This finding corroborates with what was reported in Western Kenya by Hawley et al. . This fiThe overall prevalence of malaria infection observed in this study was 53.4%. This finding is comparable to other works carried out in other malaria-endemic areas; 57.5% reported in Gabon by Bayo-Akotet et al. , 54% in P. falciparum infection occurring between the 9 and 16 weeks of gestation [Eighty-six (79.6%) women in their first trimester were highly infected than those in their second and third trimester with prevalence 46.6% (68) and 41.6% (64), respectively. The highest prevalence was obtained during the first trimester of pregnancy and decreased steadily during the second and third trimesters. This finding agrees with reports of other studies in which the peak prevalence was in weeks 10-20 of pregnancy , 38. Thiestation . In contestation . Amira restation .The current study equally revealed that the prevalence of malaria infection is more in primigravidae 66 (68.8%), followed by secundigravidae 36 (52.9%), while multigravidae had only 116 (47.5%). This finding is supported by the observation from other investigations, where primigravidae was reported to be highly susceptible to malaria infection . It is aMalaria parasitemia was more prevalent in women aged between 14-24 years (58.2%) and 25-34 years (53.2%) but was least among women from 34 years and above (41.7%). Malaria parasitaemia in relation to age showed that young pregnant women had the highest malaria prevalence rates. This finding is similar to that reported in Nigeria , 42. TheMalaria is still a major public health problem among pregnant women in Foumban Subdivision. Increase efforts should be made in order to enhance sensitization and knowledge on the importance of using ITNs during pregnancy. Furthermore, health education and other methods of malaria prevention should be made available to pregnant women as they attend antenatal clinics irrespective of the trimester of pregnancy.


Although the majority of rashes in the diaper area are caused by irritation from urine and feces, irritant diaper dermatitis; IDD, there are some less common but potentially serious cutaneous eruptions associated with systemic diseases that should not be discounted.This prospective descriptive study aimed to explore variation in cutaneous disease in the diaper area. It was conducted as a prospective descriptive study between October 2016 and November 2019 in the pediatric department of a tertiary-level hospital.Three hundred consecutive patients with rashes in the diaper area were enrolled. The most common diagnosed was IDD , followed by rashes exacerbated by the diaper  and non-diaper-related rashes .P < 0.001).Our finding suggests that when diagnosing rashes that occur in the diaper area, general pediatricians should consider, in addition to IDD, the possibility of less-common conditions. The simultaneous presence of cutaneous lesions at other sites was linked to diagnoses of systemic diseases other than IDD, ( Rashes around the diaper area are common in the pediatric population, especially among those who are diaper dependent –3. The mThis was a prospective descriptive study exploring variation in cutaneous diseases in the diaper area conducted between October 2016 and November 2019 in the pediatric department of a single tertiary-level hospital . The study was approved by the institutional review board of the Khon Kaen University Human Ethical Committee and conducted in accordance with the Declaration of Helsinki (#HE591334). The study was funded by a grant from the Khon Kaen University Faculty of Medicine in Thailand (Grant Number IN62334).All patients under 18 years of age were eligible. Consecutive patients with rashes in the diaper area who presented at the pediatric department (both in outpatient and inpatient settings) were asked to participate in the study. A total of 300 children were enrolled according to the formula for determination of sample size for estimating proportions. The estimated proportion (p) of IDD in the pediatric population was 0.25 based on the results of a previous study [Before participants were enrolled in the study, written informed consent regarding the use of all images for a medical publication was obtained from their parents or guardians.Rashes in the diaper area were diagnosed clinically by a pediatric dermatologist with some supportive investigation, such as tissue staining, blood testing, and tissue pathology, as necessary. The demographic background information collected included age, sex, general condition, initial presenting symptoms, diaper use, other underlying diseases, other sites of cutaneous eruption, and photographs of uncommon rashes in the diaper area.At the end of the study, the collected data were analyzed using STATA version 10 (StataCorp LP). Descriptive statistics – means, standard deviations (SDs), medians, and frequencies – were used to analyze the demographic data.P values < 0.05 were considered significant.A Fisher’s exact test was used for the analysis of categorical data and comparison between groups. Statistical significance was determined based on the exact approach under the Bonferroni correction. A total of 300 patients who presented with rashes in the diaper area were recruited in-to the study. Participants’ ages ranged from 2 weeks to − 168 months, with a median age of 6 months (IQR 2–29). One hundred thirty-four (44.67%) of the patients were male, and 166 (55.33%) were female. Two hundred fifteen (71.67%) were outpatient cases, and the remaining 85 (28.33%) were recruited from an inpatient setting.One hundred seventy patients with diaper rashes (56.7%) initially presented with dermatological problems, while the rest presented with problems involving other organ systems, and the diaper rashes were recognized afterward. These included gastrointestinal , infectious , respiratory , and neurological problems .The most common diagnosis was IDD , followed by intertrigo , candidiasis , eczema , and seborrheic dermatitis . Other less common cutaneous lesions in the diaper area were psoriasis, impetigo, infantile hemangioma, hand-foot-mouth disease, acrodermatitis enteropathica, staphylococcal scalded skin syndrome (SSSS), Langerhans cell histiocytosis (LCH), scabies, Henoch Sherleiin Purpura (HSP), Stevens Johnson syndrome (SJS), Lichen Sclerosus et Atrophicus (LS&A), warts, Kawasaki disease, bullous pemphigoid, and epidermolysis bullosa.The authors classified rashes around the diaper area into three types; 1) skin conditions caused by the presence of the diaper, 2) rashes exacerbated by the diaper (but not directly caused by it), and 3) eruptions present regardless of the presence of the diaper. The first was the most common type in this study, of which IDD was the only diagnosis , and the second and third were found in 101(33.67%) and 74 cases (24.67%), respectively. See Table Factors that differed significantly by type were age at onset of rash, the use of a diaper, and the presence of concurrent skin lesions at sites other than in the diaper area Table .Table 2P < 0.001: Fig. We found various types of diaper area rashes in the study population. The most common was IDD, which was found in 41.67% of participants , and those present independent of diaper use were found in 33.67 and 24.67% cases, respectively. This finding suggests that when rashes occur in the diaper area, general pediatricians should consider less -common rashes as differential diagnoses. In one case in our study, the patient was initially diagnosed with IDD before a visit to a dermatology clinic, at which the condition was diagnosed as an ulcerated hemangioma  disease was another systemic condition in the study population of which the initial presentation was diaper rash. Patients with HFM disease usually present with fever, classic cutaneous lesions of the papules or vesicular eruptions on the palms and soles, and painful vesicles on the lips and oral mucosa . In the Fig. Psoriasis can also present in the diaper area, a condition called inverse psoriasis –20. One Cutaneous candidiasis in the diaper area was the third most common diagnosis in the study population . It generally manifested as widespread erythematous plaques with discrete red satellite papules . Thus, when the patient’s chief complaint is a rash in the diaper area, complete physical examination for such lesions at other sites should be conducted to avoid misdiagnosis. Observation of cutaneous changes, complete history taking, and full repeat physical examination are also imperative in routine pediatric practice to prevent unnecessary diagnostic errors.The present study pointed out that many systemic conditions initially presented as rashes in the diaper area and may be misdiagnosed as IDD. Our study found out that the simultaneous presence of cutaneous lesions at other sites was a clue that indicated systemic diseases other than IDD, (


Plasmodium falciparum (Pf) malaria, though at a low national prevalence and heterogenous geospatial distribution. In 2015 and 2016, serosurveys were performed of children (ages 6–7 years) sampled in schools in Saut d’Eau commune  and Grand Anse department  of Haiti. Children received malaria antigen rapid diagnostic test and provided a filter paper blood sample for further laboratory analysis of the Pf histidine-rich protein 2 (HRP2) antigen, Pf DNA, and anti-Pf IgG antibodies. Prevalence of Pf infection ranged from 0.0–16.7% in 53 Saut d’Eau schools, and 0.0–23.8% in 56 Grand Anse schools. Anti-Pf antibody carriage exceeded 80% of students in some schools from both study sites. Geospatial prediction ellipses were created to indicate clustering of positive tests within the survey areas and overlay of all prediction ellipses for the different types of data revealed regions with high likelihood of active and ongoing Pf malaria transmission. The geospatial utilization of different types of Pf data can provide high confidence for spatial epidemiology of the parasite.Microscopy is the gold standard for malaria epidemiology, but laboratory and point-of-care (POC) tests detecting parasite antigen, DNA, and human antibodies against malaria have expanded this capacity. The island nation of Haiti is endemic for  Plasmodium and is found in most tropical regions throughout the world1. Elimination efforts in the 1900s and 2000s have reduced the number of countries with endemic malaria to 87, and 26 nations (as of the year 2018) are near elimination as defined by fewer than 100 indigenous cases per year2. The reduction in malaria transmission in a region is an accomplishment to be greatly celebrated, but also conveys practical challenges as a nation tries to move from a low incidence of malaria to zero malaria. With reduction of disease prevalence, funds for malaria control/elimination are often redirected to other public health efforts4. As the incidence of malaria infections decrease in a country, the capacity of health care facilities for accurate and sensitive malaria diagnosis and the availability of appropriate diagnostic tools also declines6. The concept of asymptomatic, low parasite density carriage in a population becomes much more important when planning to eliminate the entire parasite reservoir8, but current routine diagnostic tests are not ideal for identification of asymptomatic infections.Malaria is caused by parasites within the genus 9. More recently, field-deployable antigen detection tests have allowed for the widespread use of a malaria diagnostic which requires minimal user training and identifies infections with sensitivity matching or exceeding microscopy10. In the laboratory, detection of malaria parasite DNA provides a highly-sensitive measure of Plasmodium in the blood, but these assays are expensive and laborious to use on a routine basis in especially resource limited settings due to multiple processing steps and costly reagents11. Laboratory assays have recently been developed for the ultrasensitive detection of malaria antigens13. The presence of malaria antigens such as pan-Plasmodium aldolase and lactate dehydrogenase (LDH) indicate either an active infection or parasite clearance within the past 1–2 weeks14, whereas the P. falciparum histidine-rich protein 2 (HRP2) antigen is found in blood during active infection, but also for several months following parasite clearance15. Antibodies against malaria parasite antigens are a very different type of indicator for malaria exposure in that they are produced by the host adaptive immune system, and not directly by the parasite. Antibody markers can serve as indicators for historical malaria exposure, and numerous malaria antigens have now been identified that are known to induce both short- and long-lived IgG responses (months in duration to years in duration)18.To estimate malaria prevalence or transmission in an area, multiple tests are available to identify active infections, recent infections, or past exposure for individuals in the population. Historically, light microscopy for examination of blood films has been the gold standard for identification of malaria parasites with visual verification of the organism, and has been in use for over 100 yearsP. falciparum (Pf), has highly heterogeneous transmission patterns, and is at a nationwide infection prevalence of under 1%19. Results for antigen-based field diagnostics, malaria DNA assays, laboratory antigen tests, and IgG antibody detection assays were all obtained from blood samples provided by children (ages 6–7 years) participating in the study and geospatial clustering of positives was investigated. As Haiti and other countries near malaria elimination, identifying the most practical and robust tools for malaria surveillance will aid in finding the last reservoirs of Pf parasites in the population.In this study, we measure and compare multiple indicators of malaria infection or exposure from school-based surveys in two locations in Haiti conducted in conjunction with lymphatic filariasis transmission assessment surveys. Malaria in Haiti is predominantly caused by Pf exposure for these children. In Saut d’Eau commune, 4.4% of children were positive for short-term IgG (likely acquired over the past year) against Pf whereas 33.7% were positive for long-term IgG (acquired at some point in life). In comparison to Saut d’Eau, children in Grand Anse demonstrated slightly lower short-term IgG carriage (3.1%) and significantly lower long-term IgG carriage (18.7%).In total, 2,894 children were enrolled; 1,230 children from 53 Saut d’Eau schools and 1,664 children from 56 Grand Anse schools . In Saut d’Eau, RDT and PET-PCR diagnostic test positivity were associated with significantly higher short- and long-term anti-Pf IgG levels , and 20/26 (76.9%) of HRP2 antigen positives were not PET-PCR positive  measure unique markers of malaria infection or exposure, concordance among all tests was highly variable Fig. . In SautPf. In Saut d’Eau, of 53 schools, 32 (60.4%) were lacking any children who were malaria test positive at the time of enrollment, and significant clustering of positives by RDT (p < 0.05) and PET-PCR (p < 0.001) was only found in the western section of the commune  of schools having at least one antibody positive child. Significant geospatial clustering was observed for RDT (p < 0.001), long-term IgG (p < 0.001), and short-term IgG (p < 0.001) positivity – all three along the western coast of the department.In Grand Anse, 17 (25.0%) of 56 schools had children who were RDT or lab HRP2 positive Fig. . HoweverPf infection or exposure. For the Saut d’Eau commune, the PET-PCR, RDT, and long-term IgG significant prediction ellipses were all contiguous to each other or overlapping, with the PET-PCR and RDT clusters basically superimposed on each other , entomological inoculation rate , and parasite prevalence 20. These metrics have typically relied on visible identification of parasites performed by well-trained microscopists and involve a high-level of engagement and time in the field. Development and deployment of the lateral flow antigen-based malaria RDT has allowed for more pragmatic field confirmation of infection in many settings, and these tests have been reliably shown to be at least as sensitive as microscopy23. More recently, several laboratory assays have become more robust in identifying Pf biomarkers – whether these are molecules produced by the parasite during infection, or the human adaptive immune response to infection17. In both field and laboratory settings, improvement of diagnostics and assays has led to higher-throughput processing allowing a higher percentage of the sample population to be tested. The availability of multiple types of host and/or parasite biomarker data can provide better resolution in defining geospatial patterns of endemic malaria in a population and improve confidence in these data. However, it must be taken into account which data is being presented for estimates of malaria in a population as different biomarkers are measuring inherently different components of either the parasite or the host response to parasites.It is understood that no single test or assay is able to provide a complete picture of malaria transmission or prevalence in a human population. Historical metrics for malaria include: parasite rate  are geographically heterogeneous29, and accurate estimates for these patterns are deficient if only a single indicator is utilized. In this study, five different indicators of Pf infection or exposure were employed in order to estimate concordance in geospatial patterns in two separate low-transmission settings .Malaria elimination settings pose a unique challenge for defining endemic patterns of malaria transmission in a population. It has been observed that the last vestiges of malaria tend to reside in marginalized and isolated populations without adequate access to healthcare, and these persons may not exhibit expected treatment-seeking behavior even if healthcare opportunities are availablePf exposure when compared to parasite markers of active infection (antigen or DNA). Between both study sites, 26.6%  of children were positive for IgG against any of the four Pf antigens used in the immunoassay panel, whereas only 1.9%  of children were antigen or DNA positive. Detection of antibodies against Pf antigens has been utilized by many groups to estimate transmission in endemic settings, and has the advantage of detecting malaria exposure long after parasites have been cleared from the host32. The antigens chosen for the IgG detection panel in this study included two “short-term” markers  which would elicit an antibody response that would be predicted to last only a few months, and two “long-term” markers  that typically provide an IgG response lasting for years following infection33. Since the oldest children in this study were only seven years of age, positivity to either of the long-term markers would be a reliable proxy for lifetime Pf exposure. Antibody seroprevalence was found to be a useful metric for geospatial mapping, with areas of long-term IgG seropositivity found to be spatially significant for both study sites, and short-term IgG significant in Grand Anse. Since previous clinical history for these children is not known, previous Pf episodes can only be assumed from the IgG data, but long-term IgG seropositivity was much more prevalent when compared to short-term: 25.4% versus 3.7% - with only 1.5% of children being positive for short-term IgG alone. No previous clinical data is available for these children or populations, so it would be unknown if Pf occurs in outbreak cycles in the study population or is more characterized by sustained transmission.Not surprisingly, many more Haitian children were found to be positive for IgG antibody markers of previous Pf malaria, but results can also be helpful for refining epidemiological estimates. Malaria RDTs have been used for these dual purposes many times before36, and the best-in-class tests based on detection of HRP2 have been found to consistently perform well37. In this current study, 0.80%  of children were found to be RDT positive, whereas 1.7%  of children were positive by the HRP2 laboratory test. This finding is not surprising in a non-healthcare setting, as low amounts of circulating HRP2 antigen may result from a low-density infection, or could simply be antigen lingering in the blood from a previous Pf infection15. RDTs are meant to detect clinically-relevant parasite densities, and indeed many of these HRP2 + samples from RDT- children had very low concentrations of the antigen38. Among the three tests for active or very recent infection , all confirmed the low overall prevalence of Pf at these sites in Haiti. However, among these indicators and the IgG measurements, very stark differences were noted for prevalence estimates within the same study site. Numerous behavioral, environmental, and population risk factors for malaria exposure could help explain these differences and future studies should capture this information for analytical purposes.In Saut d’Eau, both DNA and RDT data demonstrated statistically significant clustering whereas in Grand Anse only RDT results showed significant geospatial clustering. The consistency of the RDT results in identifying significant clusters is encouraging since these tests are already being used in the field for identification and appropriate treatment of Pf endemicity39 as schools less than 10 km from this western cluster had radically different (lower) prevalence estimates for students with active infection and historical exposure. During these school-based surveys, demographic variables were not collected, though some information can still be inferred with knowledge of school location. All schools in this western cluster were outside of urban areas, and many were located in a low-elevation valley  based reporting as an external validation to show where Pf malaria is occurring in Haiti. The spatial estimates from this study were very closely aligned with the HF reports, especially along the western coast of Grand Anse which currently reports the highest number of Pf cases in Haiti19. The concordance of community and HF estimates, as well as the concordance among multiple field and lab Pf metrics, can provide high confidence for focusing on specific geographical areas for malaria control and elimination efforts. As these DBS samples were collected years ago, any assumptions from these geospatial analyses regarding the current transmission of a dynamic pathogen like Pf would not necessarily hold true, and new surveys would need to be performed at this time to make contemporary estimates for these areas in Haiti.These analyses are limited by the fact that this is a cross-sectional sampling design covering only a very narrow age range of the population (6–7 year olds). Malaria transmission can be very dynamic in both place and time, and given only one point in time is represented by these data, these results may not be necessarily indicative of future Assessing the concordance of separate independent indicators for malaria infection and exposure can provide robust evidence for better understanding malaria epidemiology and the geospatial overlap of independent metrics provides high confidence for detecting foci of historical and ongoing transmission. For Haiti and other countries working towards malaria elimination, combining numerous data types, as demonstrated in this study, provides more refined information on malaria that can be of potential benefit to elimination programs.27, with integration of malaria RDTs and soil-transmitted helminth microscopy of stool specimens43. Before enrollment, informed consent forms were sent home with each of the students so parent or legal guardian could read and sign for return to the study teams. Before sample collection, the informed consent forms were signed by the children’s parent, and verbal assent was given by the child participants. Fingerprick blood was collected on filter papers , dried , and packaged individually with desiccant for later laboratory analysis at the Centers for Disease Control and Prevention in Atlanta, GA. The study protocol was approved by the National Bioethics Committee of Haiti, and this activity was considered a program evaluation activity by CDC Human subjects office (#2014–256). Persons consented to future laboratory testing of DBS, and CDC laboratory staff did not have access to any personal identifiers. All methods were carried out in accordance with relevant guidelines and regulations.Samples were collected in Haiti in May 2015 from the Saut d’Eau commune and in April 2016 from the Grand Anse department as part of a lymphatic filariasis (LF) transmission assessment survey44 criteria to conduct LF TAS, with the current WHO recommendation to conduct a school-based TAS in areas where the net primary-school enrollment rate is ≥75%. Haitian school enrollment data for 2014 was utilized along with population census data to determine sampling approach employed in each EU, which are program defined. The commune of Saut d’Eau was selected as a single EU based on high baseline prevalence of LF whereas communes in the province of Grande Anse were aggregated to form a single EU based on low baseline prevalence found during initial mapping surveys45. Using the tables provided by WHO44, the target sample size and the critical cut-off threshold was determined and if the number of positive children identified fell below the critical cut-off threshold, the recommendation was made to stop mass-drug administration (MDA) for LF. Upon completion of MDA, TAS surveys were conducted to determine whether populations have reached the critical threshold of infection prevalence (<2% antigenemia), below which LF transmission is likely no longer sustainable43.Surveys were conducted in evaluation units (EUs) that had met World Health OrganizationApproximately 200 µl of blood from each child was collected via finger prick into an Heparin-coated tube. A 5 µL aliquot of blood was used for the rapid diagnostic test   and results read according to manufacturer’s recommendations. Individuals with a positive RDT result received free treatment as per the national policy in Haiti. The remainder of collected blood was applied to filter paper  and dried for a minimum of four hours to create DBS, packaged in individual baggies, and stored in a cool, dry place protected from light for less than 4 weeks until shipping to CDC in Atlanta where they were stored at −20 °C until processing.46. Briefly, the amplification of Plasmodium genus  was performed in a 20 µL reaction containing 2X TaqMan Environmental buffer 2.0  and 125 nM each of forward and reverse primers. For each sample, PET-PCR reactions were run with 2 µL of DNA template used in the PCR reaction with the following cycling parameters: initial hotstart at 95 °C for 10 min, followed by 45 cycles of denaturation at 95 °C for 10 sec, annealing at 60 °C for 40 sec. The cycle threshold (CT) values were recorded at the end of annealing step and a positive CT value was considered below 40.0. All assays were performed using a Strategene Mx3000P . To extrapolate an estimated parasite density  from a CT value, a standard curve was made using extracted DNA from the 3D7 culture strain previously quantified by microscopy46.DNA was extracted from DBS by using the QIAamp DNA Mini Kit  as recommended by the manufacturer. Briefly, a DBS tab  was placed into a 1.5 mL tube and processed according to instructions. The DNA was eluted in 150 µL of elution buffer and stored at −20 °C until use. All Saut d’Eau samples were screened using the multiplex PET-PCR assay as previously describedE. coli extract to prevent non-specific binding) to a final dilution of 1:20×. The presence and quantification of IgG32 and HRP2 antigen38 was performed with methodology similar to that described previously using the bead-based Luminex based MAGPIX platform .A single DBS tab (10 µL whole blood) was rehydrated in a blocking buffer  0.5% polyvinylpyrrolidine (SigmaAldrich), 0.1% casein , 0.5% BSA (SigmaAldrich), 0.3% Tween-20, 0.05% sodium azide, and 0.01% P. falciparum HRP2 IgG) at 20 µg per 12.5×106 beads. Biotinylated detection antibodies  were prepared by Thermo Scientific EZ-Link Micro Sulfo-NHS-Biotinylation Kit (ThermoFisher) according to the manufacturer’s protocol and stored at 4 °C at a final concentration of 1 mg/mL. All reagents were diluted in buffer containing PBS, 0.05% Tween20, 0.5% Bovine Serum Albumin (SigmaAldrich), and 0.02% NaN3. Samples (50 µL of 1:20x dilution whole blood) were incubated with anti-HRP2 coated beads for 90 min at room temperature under gentle shaking protected from light in MultiScreen-BV filter plates . After three washes , beads were incubated with 50 µL biotinylated detection antibody (1:500) for 45 min with same incubation conditions as above. After three washes, 50 µL streptavidin-phycoerythrin  was added to all wells (1:250x of 1 mg/mL) for a 30 min incubation. After three washes, samples beads were incubated with 50 µL reagent buffer for 30 min, washed once, and resuspended in 100 µL PBS. Assay plates were shaken briefly and read on a Bio-Plex 200 machine (Bio-Rad) by generating the median fluorescence intensity (MFI) for 50 beads. The final measure, denoted as MFI-bg, was reported by subtracting MFI values from beads on each plate only exposed to sample diluent during the sample incubation step. The MFI-bg threshold for a true positive HRP2 assay signal was ascertained if the sample MFI-bg was higher than the mean + 3 SD of the MFI-bg signal of a panel of known negative DBS samples. To translate between a MFI-bg value and antigen concentration for classified positive samples, equations for a standard curve of recombinant HRP2 were calculated. Recombinant PfHRP2 antigen was provided by Microcoat Biotechnologie GmbH, and lyophilized preparations were rehydrated according to the manufacturer’s instructions.For the HRP2 detection assay, a carboxylated bead set  was coated by the EDC/Sulfo-NHS intermediate reaction with anti-HRP2 antibodies 5 repeat , liver stage antigen 1 Pl1043 epitope , merozoite surface protein 1 19kD fragment , and apical membrane antigen 1 N-terminal region .Four separate bead regions (Bio-Plex) were coupled with malaria antigens for IgG capture and subsequent detection. The antigens in the multiplex panel have all been reported on before4, both produced by Southern Biotech, Birmingham, AL) for 45 min with same incubation conditions as above. After three washes, 50 µL streptavidin-phycoerythrin (Invitrogen) was added to all wells (1:250x of 1 mg/mL) for a 30 min incubation. After three washes, samples beads were incubated with 50 µL reagent buffer for 30 min, washed once, and resuspended in 100 µL PBS. Assay plates were briefly shaken and read on a Bio-Plex 200 machine (Bio-Rad) by generating the median fluorescence intensity (MFI) for 50 beads. The final measure, denoted as MFI-bg, was reported by subtracting MFI values from beads on each plate only exposed to sample diluent during the sample incubation step. The MFI-bg threshold for true positive IgG assay signal was ascertained if the sample MFI-bg was higher than the mean + 3 SD of the MFI-bg signal of a panel of known negative DBS samples.Samples (50 µL of 1:200x dilution whole blood) were incubated with beads for 90 min at room temperature under gentle shaking protected from light in MultiScreen-BV filter plates (MilliporeSigma). After three washes , beads were incubated with 50 µL biotinylated detection antibody .Cluster analysis was performed using the software SaTScan (Version 9.6) which employs Kulldorff’s spatial scan statistic. The software compared multiple moving windows with variable radii ranging from the smallest observed distance to a pre-determined upper bound across the study area. Likelihood ratios were used to test whether there was an elevated seropositivity inside each window compared to outside the windowt-test of log-transformed assay signal values by the PROC TTEST procedure in SAS v9.4 . Regression comparing PET-PCR to HRP2 antigen concentration was performed in Excel.Significant differences in in IgG antibody signal by PET-PCR, RDT, or HRP2 antigen test positivity was determined by The findings and conclusions in this report are those of the authors and do not necessarily represent the official position of the CDC.Supplementary Information.


P. falciparum malaria (B50.8 ICD 10). Objective. The aim of this study was to improve the intensive care treatment for severe and complicated P. falciparum malaria. The treatment strategy implemented was aimed at preventing ischaemia-reperfusion injury to organs, as well as haemorrhagic complications. The ICU Case Management Protocol set up indications for transferring patients to the ICU which provide preventive  application of extracorporeal hemocorrection methods (continuous venous-venous hemodiafiltration and plasmapheresis in a plasma exchange mode) and mechanical ventilation under a medically induced coma, given impaired consciousness as the initial symptom of patients. Results. Successful treatment outcome in a majority of the patients (93.8%), shorter ICU length of stay (6.67 ± 1.9 days as compared to 94 ± 1.6 before introduction of the protocol), a median parasite clearance time of 37.50 hours (95% CI 36.21–38.18), and a reduced mortality rate from 29.1% to 6.25% support the efficacy of the ICU protocol in managing severe and complicated P. falciparum malaria.This study analyses the intensive care treatment of 48 patients admitted to the Intensive Care Unit (ICU) at the Infectious Diseases Clinical Hospital No. 2, Moscow, Russia, between 2007 and 2019, with a severe and complicated form of  P. falciparum malaria from high endemic countries in Africa, Southeast Asia, and in the recent years from South America are recorded annually in the Russian Federation [Imported cases of deration .Anopheles mosquitoes are nonsusceptible to P. falciparum [P. falciparum transmission by local mosquitoes for over 40 years. There were 1086 confirmed cases of P. falciparum malaria in the Russian Federation between 2000 and 2019, of which 44 patients died (4%). Moscow city and the Moscow region had the highest incidence of imported cases with 453 reported cases and 13 deaths (2.9%) between 2009 and 2019. Factors found to be associated with severe and complicated P. falciparum malaria were absence of chemoprophylaxis by travellers to endemic regions, lack of information on travel history, healthcare delay, and management and diagnostic errors. In multiple cases, medical practitioners implement conventional antimalarial medication without considering the risk of a drug-resistant P. falciparum in endemic regions where travellers may have visited [P. falciparum malaria are nonimmune Russian citizens (travellers) which explains the rapid development of severe and complicated forms of the disease. The aim of this study was to improve the intensive care treatment for patients with severe and complicated Р. falciparum malaria so as to prevent fatal outcomes.Review of data on susceptibility of mosquitoes in the USSR to imported strains of malaria parasites revealed that local lciparum . This faP. falciparum malaria admitted to the ICU at the Infectious Diseases Clinical Hospital No. 2, Moscow city, in the period from 2007 to 2019. These patients had been managed with a protocol for severe and complicated P. falciparum malaria which was developed at the ICU. The ICU Management Protocol for severe and complicated P. falciparum malaria consists of modern methods of extracorporeal hemocorrection (continuous venous-venous hemodiafiltration and plasmapheresis in a plasma exchange mode) and mechanical ventilation under a medically induced coma. A control group was studied to determine the efficacy of the ICU Protocol. This group consisted of 24 patients with severe and complicated P. falciparum malaria who were managed without the ICU Protocol because they were admitted before the Protocol was developed (period from 2003 to 2007). All patients had received conventional antimalarial medications. The characteristics of the two groups are presented in We analyzed the management of 48 patients with severe and complicated All patients were evaluated under the WHO criteria for severe malaria . It was The treatment strategy was to prevent ischaemia-reperfusion injury to organs, as well as to prevent haemorrhagic complications. After admission to the ICU, central venous catheterization was performed for parenteral administration of drugs. Ischemia and hypoxia have been implicated in cerebral malaria (CM); therefore, oxygen delivery was executed, and hydration was maintained. The ICU Protocol included the utilization of extracorporeal hemocorrection and mechanical lung ventilation. An indication for these procedures was late admission (>5 days from the illness onset), regardless of the level of parasitaemia. Patients showing signs of cerebral malaria  were a strong indicator for mechanical ventilation. This procedure was executed under anaesthesia-inducing drugs (propofol + midazolam). Mechanical ventilation was performed in 20 (41.6%) patients in the main group who presented with altered mental status and impaired consciousness. Extracorporeal hemocorrection involved the use of continuous veno-venous hemodiafiltration and plasmapheresis in a plasma exchange mode. A second large bore two-way IV set was inserted into the femoral vein for extracorporeal hemocorrection procedures. Continuous veno-venous hemofiltration was performed in 37 (77.1%) patients of the main group, using the PrismaFlex system or multiFiltrate devices .Plasmapheresis was introduced in the Protocol in 2014 and was performed in 14 patients of the main group (29.2%). One of the main indications for plasmapheresis was high parasitaemia and metabolic disorders. Intravenous (IV) infusion of 20% mannitol (0.2 g/kg body weight) plus 20 mg furosemide was indicated for cerebral oedema. During the process of fluid infusion, plasma and urine osmolarity, plasma acid-base and electrolyte equilibrium, plasma albumin, and central venous pressure were monitored. The volume of parenteral fluid infused was determined by the volume of enteral nutrition and renal and extrarenal loss. Blood glucose was monitored every 4 hours. Hypotonic solutions and dextrans were not utilized. We managed patients with antioxidants and antihypoxants and implemented prophylactic infusion of fresh frozen plasma to prevent haemorrhagic complications. Clinical and laboratory monitoring of the patients' condition included a complex of clinical biochemistry blood tests, urine test, and other biological specimen and the use of instrumental devices based on indications, echocardiography, chest radiograph, ultrasound of the abdominal, and retroperitoneal and pleural cavities.P. falciparum malaria in nonendemic regions are common, and there is a high risk of complication in nonimmune individuals from these regions. P. falciparum malaria parasites are increasingly resistant to antimalarial drugs; therefore, besides provision of antimalarial drugs, medical experts in nonendemic countries implement strategies to improve intensive care and utilize adjuvant therapies to reduce mortality [P. falciparum malaria was updated in 2014 after the introduction of plasmapheresis for intensive care management, given the issue that life-threatening state and complications could occur even after elimination of malaria parasites. Nevertheless, it should be noted that any therapeutic approach based on the use of antimalarial drugs and interventions to correct impaired functions of organs and systems does not always ensure a successful treatment of severe and complicated P. falciparum malaria. We evaluated the efficacy of extracorporeal hemocorrection methods in severe malaria by determining the asexual parasite clearance. The parasite clearance estimator (PCE) developed by the Worldwide Antimalarial Resistance Network (WWARN) seemed to be an accurate and reliable method [p < 0.05 .Imported cases of severe ortality , 8–13. Portality , 13. Frop < 0.05 . This reP. falciparum malaria infection influences blood coagulation by various interacting pathobiological mechanisms, the most important being the overwhelming response of the host to sepsis resulting in a cytokine storm. In addition, the parasite infects the red cells leading to changes in the red cell phospholipid composition which supports blood coagulation. Red cells infected with P. falciparum also adhere to the deeper tissue capillary endothelium leading to profound damage to endothelial cells leading to further activation. This results in widespread consumption of platelets and activation of blood coagulation which, at times, culminates in a clinically and pathologically detectable disseminated intravascular coagulation (DIC). Manifestations of blood coagulation disorders are recorded in about 5% of patients with severe P. falciparum malaria. We recorded 3 cases of blood coagulation disorders in the main group, 2 of which had died. The transfusion of whole blood and other blood components, red cells, and platelet concentrate remains the most effective treatment for blood coagulation disorders. Thirty-seven (77.1%) patients received plasma and other blood components, 9 patients (18.8%) received transfusion of whole blood and/or RBC, and 6 (12.5%) received platelet concentrate. The protocol was aimed at restoring impaired homeostasis, and the theoretical insight of the procedures involves the removal of a wide variety of biologically active substances . Numerous studies have implicated a dysregulated immune response and cytokine balance to severe P. falciparum malaria [ malaria –17, and Eventually, 45 out of 48 patients recovered fully (93.75%) in the main group. Three deaths were recorded in the main group (6.25%), 7 deaths recorded in the control group (29.1%) with 14 fully recovery, the main group had a shorter ICU length of stay of 6.67 ± 1.9 days compared to (9.4 ± 1.6 days) the control group. These outcomes demonstrate the efficacy of the approach implemented.P. falciparum malaria managed under the ICU Management Protocol for severe and complicated P. falciparum malaria: A 49-year-old man presented to our hospital at the ICU on March 30, 2020, with persistent fever and fatigue for 10 days. A day before, a local hospital had identified P. falciparum malaria parasites in his blood smear. The patient noted that a high fever (38.5°C) began 4 days before his return from Zimbabwe on the 21st of March. He did not take antimalarial prophylaxis in Africa. His condition was considered severe with a score of 13 on the Glasgow Coma Scale (GCS). Vital signs were as follows: a temperature of 38.9°C, heart rate of 120 beats/min, blood pressure of 100/70 mmHg, and respiration rate of 28 breaths/min. The patient appeared acutely ill with generalized weakness and drowsiness. His abdomen was soft and distended. The neurological examination was normal although he faintly responded to verbal commands. On examination of blood smears, parasite load for P. falciparum was 1846720 mcL at admission and was diagnosed with severe P. falciparum malaria: cerebral forms and multiorgan failure. The severity of the patient's condition is due his nonhistory of malaria (nonimmune), late admission, high parasitaemia, and clinical presentation at admission. Laboratory results were as follows: complete blood count revealed a white blood cell (WBC) count of 9.2 per microliters of blood, haemoglobin of 14.2 g/dL, and a platelet count of 11 per microliter of blood. Urinalysis showed protein (+) in the urine. Hepatorenal impairment was present with an aspartate aminotransferase level of 113 IU/L, alanine aminotransferase level of 65 IU/L, total bilirubin (T-bili) of 94 micromol/L, direct bilirubin (D-bilirubin) of 41 micromol/L, blood urea nitrogen (BUN) of 18.8 micromol/L, and creatinine of 156 micromol/L. An ultrasound of the abdomen demonstrated hepatosplenomegaly, haemangiomas of the liver, and deformation of the gallbladder. After admission to the ICU, central venous catheterization was performed for parenteral administration of medications. He was intubated and ventilated due to cerebral manifestations. From the first hours of admission at the ICU, antimalarial therapy (quinine 1 g/daily) and 0.2 g of doxycycline daily were initiated. He was monitored every day for thrombocytopenia. His central venous pressure, acid-base, and hydration status were also monitored. Infusion of fresh frozen plasma and platelet transfusion were initiated. Given the patient's severe status, we initiated our protocol for adjuvant treatment of severe malaria. Our patient required 8 sessions of continuous veno-venous hemodiafiltration (AN69 polysulfone Baxter membrane) from 03/31/2020 to 04/07/20 and 4 sessions of plasmapheresis in a plasma exchange mode (Plasma filter membrane PFM-500) from 03/31/2020 to 04/03/20. After 3 days of quinine intervention, anuria (nonpassage of urine) and symptoms of acute kidney injury increased. Laboratory examination revealed urea nitrogen (BUN) of 53.7 micromol/L, creatinine of 703 micromol/L, serum potassium elevation of 6.4 mmol/L, and haemoglobin of 14.2 g/dL. Echocardiography showed pericardial effusion. An ultrasound of the abdomen demonstrated oedema of the surrounding gallbladder tissue. We ceased quinine treatment due to possible renal failure after erythrocyte haemolysis and switched to artesunate. After 5 days of treatment at the ICU, his peripheral blood smears did not contain any ring-form trophozoites anymore. In the absence of blood parasites, it was decided to stop artesunate treatment and switch to 80 mg coartem. Our patient remained on ventilator therapy for 8 days and developed ventilator-associated pneumonia (VAP) for which he was treated with meropenem for 10 days. By the 10th day of illness, the level of urea and creatinine stabilized, and, given his stable state, he was transferred from the ICU to the infectious general ward for further treatment and observation. By the 19th day of stay at the hospital, the patient's clinical outcome improved and his appetite returned to normal. There were no complications from malarial infection. Haemoglobin, bilirubin, and liver enzyme levels returned to normal. He was discharged after 21 days.We present a case of severe P. falciparum malaria which has been in use for over 10 years together with the WHO guidelines (2010 and 2015) for treatment and management of severe malaria cases. This protocol provides preventive  application of extracorporeal hemocorrection methods (based on continuous venous-venous hemodiafiltration and plasmapheresis in a plasma exchange mode) and mechanical ventilation under a medically induced coma, given impaired consciousness as the initial symptom of most patients admitted to the ICU. After implementing the protocol, 93.75% of patients with severe and complicated P. falciparum malaria recovered and had an average ICU stay of 6.67 ± 1.9 days. The mortality rate of severe P. falciparum malaria was reduced to 6.25% from 29.1% before the protocol was introduced which largely demonstrates its efficacy. The procedures and interventions involved were introduced with the key intention of saving the lives of patients, though quite expensive. Therefore, it is not considered in financing programs and measures to combat malaria. The outlined ICU Protocol would gain the attention of medical practitioners, in terms of organisation (logistics). Cases of severe malaria can be avoided by adhering to general recommendations and measures which include observing chemoprophylaxis when traveling to endemic regions, prompt diagnosis, and adequate treatment of patients. The authors note the need for systematic training and education of medical practitioners including acquiring the knowledge of drug-resistant parasites. Health recommendations and general travel advice for international travellers to endemic regions should be pursued. Another vital strategy which the authors support is to increase accessibility and enforce license of effective first-line antimalarial drugs.A combination of conventional antimalarial drugs and adjuvant intensive therapeutic interventions in severe malaria cases could reduce mortality cases. It is on this note that the authors developed and implemented a protocol for managing severe cases of


Plasmodium infections are co-endemic with infections caused by other agents of acute febrile illnesses, such as dengue virus (DENV), chikungunya virus, Leptospira spp., and Orientia tsutsugamushi. However, co-infections may influence disease severity, treatment outcomes, and development of drug resistance. When we analyzed cases of acute febrile illness at the All India Institute of Medical Sciences, New Delhi, India, from July 2017 through September 2018, we found that most patients with malaria harbored co-infections (Plasmodium mixed species and other pathogens). DENV was the most common malaria co-infection . DENV serotype 4 was associated with mild malaria, and Leptospira was associated with severe malaria. We also found the presence of P. knowlesi in our study population. Therefore, in areas with a large number of severe malaria cases, diagnostic screening for all 4 DENV serotypes, Leptospira, and all Plasmodium species should be performed.  Orientia tsutsugamushi, Leptospira, and Salmonella enterica serovar Typhi), parasitic (protozoans of the apicomplexa family), or viral   constitute a group of infections with similar manifestations, such as fever, malaise, body aches, chills, hepatic and renal dysfunction, and central nervous system effects. The causative agents of AFI can be bacterial  from the Department of Medicine at All India Institute of Medical Sciences from July 2017 through September 2018. Every admitted consenting AFI patient was tested by PCR for all 5 Plasmodium species , DENV, CHIKV, O. tsutsugamushi, and Leptospira. The study was approved by the institute research ethics committee .For our prospective study, we recruited patients with an AFI . Typhoid testing was not conducted for patients with no abdominal pain or diarrhea. None of the patients recruited for this study showed indications for typhoid testing. All patient data were anonymized to protect confidentiality. Blood samples were collected and subjected to microscopy, rapid diagnostic testing, and PCR analysis for all 5 pathogens . The rapid diagnostic test detects antigens specific to histidine-rich protein II from P. falciparum and pan-Plasmodium lactate dehydrogenase from P. vivax, P. malariae, or P. ovale.For microscopic examinations, we used peripheral blood smears (Giemsa-stained thick and thin smears) and a 3-band rapid diagnostic test kit . These guidelines define severe malaria as creatinine level >3 mg/dL, bilirubin level >3 mg/dL, bicarbonate level <15 mmol/L, hemoglobin level <7 g/dL for adults and <5 g/dL for children, parasite count 10%, hypoglycemia <2.2 mM, substantial bleeding, impaired consciousness, shock, prostration , multiple convulsions, and pulmonary edema)  according to the manufacturer’s instructions. To detect DENV and CHIKV, we extracted RNA from TRIzol by using the isopropanol method, and we synthesized complementary DNA from RNA by using a Verso cDNA Synthesis Kit  according to the manufacturer’s recommendations. We analyzed all samples for the presence of all 5 human Plasmodium species, O. tsutsugamushi, Leptospira, DENV, and CHIKV. All samples were also subjected to microscopy and rapid diagnostic testing (for PfHRP2 and PvLDH genes) for malaria diagnosis. The diagnosis of DENV and its serotypes was conducted by using serotype-specific PCR primers. The presence of other infectious agents, such as O.tsutsugamushi, Leptospira, and CHIKV was detected by PCR , as described previously (http://www.indianremotesensing.com/2017/01/Download-India-shapefile-with-kashmir.html) and generated the final image by using Microsoft PowerPoint (https://www.microsoft.com) to map each patient to their local area. In addition, the 12 patients with P. knowlesi infection were asked to answer questions about time of malaria infection (as recorded in our dataset), travel outside India in 2 years preceding the malaria infection, visits from abroad by friends/relatives, and any previous malaria infections (possibility of recurrence/relapse).We were able to retrieve location data for 82 patients. We constructed a map of India based on the official maps provided by the Survey of India  for each co-infection by using Stata version 15.0 statistical software (https://www.stata.com). We considered p<0.05 to be statistically significant. We created a patient baseline characteristics table by using the R version 3.4.3 package tableone  and age as means. We used χPlasmodium mixed-species infections in the 66 Plasmodium-positive samples (P. vivax accounted for most (76%) (50/66) infections, whereas P. falciparum accounted for 35% (23/66). P. knowlesi was detected in 18% (12/66) of infections  samples indicated a DENV co-infection with or without other co-infecting pathogens, and 29 (44%) indicated exclusive Plasmodium/DENV co-infections. Plasmodium co-infections with bacteria were found for 16 (25%) patients: Leptospira infections for 11 (17%) of the 66 and O. tsutsugamushi for 5 (8%) (From the 66 r 5 (8%) .P. knowlesi infection originated from Delhi, Haryana, Uttar Pradesh, and West Bengal. Most patients with dengue infections originated from Delhi and Uttar Pradesh. Of the 12 patients with P. knowlesi infection, 5 had not traveled abroad or had direct contact with any visitors from abroad for at least 2 years before admission. No information was available for the remaining 7 patients , leukocyte counts (10.53 vs. 6.07) × 103 cells/μL), and creatinine levels (3.37 vs. 0.90). Each group contained 26 male patients; mean age for severe malaria patients was 28 years and for mild malaria patients was 32 years.Detailed hematologic and biochemical parameters for all patients were retrieved from medical records  Table 4.Leptospira  or O. tsutsugamushi  was associated with severe malaria. P. vivax or P. knowlesi monoinfection was also associated with severe malaria   was associated with mild malaria , whereas infection with 0.9–7.2) . Other cseverity  Table 5.Plasmodium species by microscopy (8 positive results), rapid diagnostic testing (26 positive), and PCR (66 positive)  . Almost fections . The diafections  Table 6.Plasmodium species was P. vivax and malaria/DENV co-infections predominated. A high number of severe malaria cases reported to the institute are from northern India. Among the 5 Plasmodium species known to infect humans, in our study population we detected P. falciparum, P. vivax, and P. knowlesi but found no evidence of P. malariae or P. ovale. Most AFI patients in this study originated from northern India across the states of Rajasthan, Haryana, Punjab, Delhi, Uttar Pradesh, Bihar, and West Bengal. The burden of co-infecting pathogens in patients with malaria was revealed by a combination of complete blood work  and in-depth molecular assays (PCR amplification of Plasmodium species–specific genes followed by Sanger sequencing). We found a very high percentage of Plasmodium/DENV co-infections in our study population. This finding can be partly attributed to the highly sensitive PCR diagnostic methods used.Among patients hospitalized with AFI at the All India Institute of Medical Sciences during July 2017–September 2018, the major circulating Plasmodium/CHIKV co-infections spanning 7 geographic regions  showed that DENV/Plasmodium co-infections have been reported in 19 countries, including India; DENV/CHIKV co-infections have been reported in 24 countries including India; CHIKV/Plasmodium co-infections have been reported in 6 countries with only a single co-infection reported from India; and DENV/CHIKV/Plasmodium co-infections have been reported in 3 countries , because it has not been widely reported. However, India is known to harbor both the potential vector for P. knowlesi, the Anopheles dirus mosquito, as well as the reservoir, pig-tailed macaques (Macaca nemestrina), thereby making India a potential ecosystem for the proliferation of this zoonotic Plasmodium species (Macaca mulatta macaques and related species have recently expanded in northern India, particularly in the state of Uttar Pradesh, which may explain the appearance of P. knowlesi in our study population representative of these areas, whereas it was not reported earlier (P. falciparum parasites in monkey populations from India, indicating freely occurring undetected zoonotic transfer of the malaria parasites across reservoirs and hosts. Therefore, healthcare workers and national programs should incorporate all species of malaria parasites known to infect humans, in their diagnostic portfolio.To assess whether the infections originated locally, we surveyed the P. knowlesi infections, co-infections and mixed Plasmodium infections were highly prevalent in patients with malaria, Plasmodium/DENV co-infections were the most common co-occurring pathogens in our study population, P. knowlesi infections were present in India, Plasmodium/DENV4 co-infections were associated with mild malaria, and Plasmodium/Leptospira infections were associated with severe malaria. Although the ORs support the above findings, the 95% CIs for these associations were wide. CIs reflect the uncertainty of the estimated effect, and wider intervals suggest greater uncertainty. The wide 95% CIs in this study suggest that although trends were observed, additional data points are needed to determine the effect size of these associations. Wider prevalence studies investigating malaria co-infections are needed.In conclusion, our study clearly showed that microscopy and rapid diagnostic testing gave false-negative results for most mixed-species infections and completely missed Plasmodium reservoir, contributing to almost 4% of malaria-related deaths globally. Therefore, to make malaria elimination possible, we offer 2 recommendations based on our observations in this study, particularly for tertiary healthcare centers or centers where the burden of severe malaria cases is high. First, malaria elimination efforts will need to include strategies for malaria elimination in humans as well as animal reservoirs. Second, efforts toward the development of novel diagnostics for malaria must be renewed, and AFI diagnoses must include screening for all 5 Plasmodium species, Leptospira, and all 4 DENV serotypes.The government of India has recently declared a goal of malaria elimination by 2030, which will be a major step toward global malaria eradication because India serves as a major leptospira co-infections with malaria severity.Supplementary methods and results for study of association of dengue virus and