Plasmodium vivax, and the serological responses to P. vivax and Plasmodium falciparum antigens that reflect past or present exposure. A focus has been studying the pathogenesis of severe malaria caused by P. falciparum through magnetic resonance imaging of cerebral malaria patients. Here we provide a snapshot of some of the basic and applied research the CSCMi has undertaken over the past 12 years and indicate the further research and/or clinical and translational impact these studies have had.The Center for the Study of Complex Malaria in India (CSCMi) is one of 10 International Centers of Excellence in Malaria Research funded by the National Institutes of Health since 2010. The Center combines innovative research with capacity building and technology transfer to undertake studies with clinical and translational impact that will move malaria control in India toward the ultimate goal of malaria elimination/eradication. A key element of each research site in the four states of India  has been undertaking community- and clinic-based epidemiology projects to characterize the burden of malaria in the region. Demographic and clinical data and samples collected during these studies have been used in downstream projects on, for example, the widespread use of mosquito repellants, the population genomics of  Plasmodium falciparum is the dominant species and is transmitted by the major vector Anopheles gambiae, malaria in India can be caused by at least four parasite species , reflect coinfections of two or more species or genotypes, and can be transmitted by at least six Anopheles species, including An. stephensi, An. fluviatilis, An. culicifacies, An. minimus, An. baimaii, and An. annularis. The overall goal of CSCMi is to address major gaps in our understanding of such “complex malaria” and to elucidate how it affects the epidemiology, transmission, drug resistance, parasite population genomics, and pathogenesis of malaria in India. Key questions that we have attempted to answer include the following: What is the eco-epidemiology and transmission of complex malaria at our field sites and how is it changing? What is the role of environmental conditions in determining malaria transmission intensity in different eco-epidemiological contexts? And how genetically diverse is P. vivax and how does this compare with P. falciparum?The Center for the Study of Complex Malaria in India (CSCMi), one of 10 International Centers of Excellence in Malaria Research (ICEMRs) located in malaria-endemic regions of the world, was launched in 2010.P. falciparum, a drop in incidence may be accompanied by a rise in the proportion of acute cases,P. falciparum/P. vivax coendemic areas of the world, where the burden of the former has been reduced by control programs, a rise in the proportion of malaria caused by the latter has been reported.P. vivax infection challenge this assumption.Twelve years later our research questions remain highly relevant. According to the WHO, India has had the largest absolute reductions in malaria cases in Southeast Asia, from 19.7 million in 2000 to 5.6 million in 2019, although it still contributed 86% of malaria cases in the region that year.The multidisciplinary partnership between Indian and international institutions under the umbrella of the CSCMi has advanced both basic and translational research, from updating epidemiology in malaria endemic states with distinct ecological and ethnicity profiles, to generating the first whole genome sequences of Indian malaria parasites and deciphering molecular mechanisms leading to the development of cerebral malaria. The dynamic and adaptive design of the CSCMi has enabled rapid responses to new challenges and allowed the development of spinoff projects such as a study of the etiology of nonmalaria febrile illness  in a hospital setting in Rourkela, Odisha.P. falciparum in forest/riparian ecology), in the south , and in the west   ; see Dasciparum) . Due to Our initial projects elucidated the baseline epidemiology of regions in four states of India  that differ by climate, ecology, and ethnic make-up , through,Plasmodium infections are increasingly associated with few symptoms and low parasite biomass, making diagnosis challenging and underscoring the need for more sensitive diagnostic tests at the point of care. Recently, it was proposed that facilities created for COVID-19 diagnosis—including PCR assays in field-friendly formats deployed across the country—can easily be coopted and harnessed for malaria diagnosis.Plasmodium infections. We found that addition of the symptoms “headache,” “aches,” and “chills” to “fever” improved the case definition of symptomatic malaria. Our findings indicate that malaria and asymptomatic infections differ by region in India, indicating that eliminating malaria will require localized approaches.A major revelation of our cross-sectional studies in Gujarat, Odisha, and Tamil Nadu during 2012–2015 has been the number of asymptomatic and submicroscopic infections. For example, 71% of infections in cross-sectional surveys in Chennai, Tamil Nadu, were asymptomatic, and 71% of infections were submicroscopic.Our household census data in Gujarat, Odisha, and Tamil Nadu showed that the use of mosquito repellents such as coils, vaporizers, and mats was common and influenced by education level and socioeconomic status, but was not consistently associated with a reduction in malaria in our cross-sectional and clinic data.P. vivax used PCR to detect Plasmodium parasites in 131 contacts of 20 index cases  and 868 contacts of 18 index cases , a strategy that identified only four new infections at the latter site. RCD thus proved to be a labor-intensive strategy that was not useful at our vivax endemic sites.P. vivax infection that could indicate hypnozoite carriers to be targeted for treatment with antihypnozoite drugs .P. vivax and P. falciparum at all four of our sites in India, using either ∼1,000 Plasmodium antigens spotted on microarray chipsPlasmodium antigens that reflect past or present exposure.Plasmodium antigens and P. falciparum antigens associated with asymptomatic infections.Plasmodium antigens to classify exposure as either recent or long term in studies investigating a recent P. vivax outbreak in Jaintia Hills, Meghalaya, and as part of the malaria camps effectiveness trial mentioned earlier.We have piloted novel strategies for malaria surveillance and elimination at several of our sites. In 2014, our reactive case detection (RCD) studies at two urban areas with low prevalence of mainly Anopheles species in the same villages as our epidemiology studies, quantifying the role of environmental conditions in determining malaria transmission intensity, and identifying novel mutations associated with insecticide resistance that key Indian Anopheles vectors have developed.Our transmission and vector studies in Tamil Nadu, Odisha, and Meghalaya have included surveillance projects of Anopheles mosquitoes in India, an important component of the CSCMi has been capturing Anopheles adults using CDC light traps and aspirators, accompanied by morphological and molecular classification, salivary gland and midgut dissections for malaria parasite detection, and blood meal analysis. One surveillance study in 15 villages in Odisha during 2012–2013 confirmed An. culicifacies and An. fluviatilis as the major vectors in this area, with greater densities found in cattle sheds compared with human dwellings.An. fluviatilis S-type to the more zoophilic T-type with a preference of cattle sheds over human dwellings (as described in other studies)An. stephensi as greatly preferring cattle sheds to human dwellings for both resting and feeding, and those found in human dwellings greatly preferring thatched structures.Anopheles subpictus, not previously considered a human malaria vector in this city.Anopheles species, some of which are known malaria vectors, including Anopheles jeyporiensis.An. baimaii and An. minimus corroborated recent reports from northeast India that these forest-associated mosquito species are in decline.Given the diversity of An. stephensi, leading us to recommend directing intervention efforts to that habitat.An. stephensi immature density, an indicator/predictor of vector breeding.An. stephensi, and dawn is the optimal time to collect and estimate its densities.We monitored temperatures in a range of indoor and outdoor resting habitats of mosquitos in two urban slum sites at our Chennai field site because standard estimates of environmental temperature derived from the local weather stations do not provide realistic measures of temperatures within actual transmission environments, and even small differences in mean temperatures or diurnal temperature ranges could lead to large variations in mosquito and/or parasite life history traits that determine transmission intensity.kdr) in Indian mosquito species due to mutations in the voltage-gated sodium channel gene that confers resistance against DDT and the pyrethroid group of insecticides. We reported two alternate point mutations leading to the same amino acid substitution (L1014F) in An. subpictus and developed a new assay that detected the mutations at high frequency (82%) in the Indian study area.kdr mutations, L1014F and L101S, in An. stephensiAn. culicifaciesAn. stephensi under laboratory conditions and identified tandem duplication of a genomic segment of the GSTe (epsilon class of glutathione S-transferases) gene array encoding GSTe2 and GSTe4, leading to increased transcription of these two genes. The duplication event also led to the diversification of these two genes, resulting in two paralogues of GSTe2 and three paralogues of GSTe4 being present in a single mosquito.Our initial studies involved detection of “knockdown resistance”  technologyP. falciparum drug resistance genes .PfK13 associated with artemisinin resistance, as well as heterozygous loci containing allelic variants, indicative of mixtures of resistant and sensitive clones in the same individual. Any marker gene can be swapped into the panel, making this “amplicon-seq” method potentially suitable for studies of, for example, P. vivax drug resistance or the number of Plasmodium genotypes in an infection (“complexity of infection”).We set up an NGS facility at the ICMR/National Institute for Malaria Research in DelhiP. vivax and P. falciparum reference genomes from India.P. vivax strains from India, Latin America, North Korea, and Mauritania with six comparator P. falciparum lines revealed twice as much genetic diversity in P. vivax, suggesting a more stable and older association of this species with humans than for P. falciparum. In addition, the greater genetic diversity and gene family variability that we found suggests an increased capacity for functional variation in the global P. vivax population. We concluded that P. vivax malaria will likely be the more difficult parasite to eliminate.One of our major achievements has been to generate the first P. vivax clinical isolates from five of the ICEMRs,P. vivax selective sweeps of two drug resistance-associated genes PvDHR and PvDHPS. Other signals of natural selection that we identified suggest that P. vivax is adapting to regional differences in the human host and mosquito vector. The several Indian P. vivax genomes from our Chennai site showed significant admixture with isolates from Africa and support the hypothesis that contemporary African and South Asian P. vivax populations are genetically similar, suggesting that the latter may have genetically mingled with European (“New World”) lineages during the colonial era.We generated whole genome sequences of ∼180 global P. vivax and P. falciparum, which has allowed us to investigate severe malaria caused by both species in different eco-epidemiological settings.India is endemic for ,Severe falciparum malaria is a complex disease with a wide spectrum of manifestations,P. falciparum-endemic setting of Odisha, where both pediatric and adult CM cases occur, leveraged the availability of a 1.5-Tesla MRI scanner at Ispat General Hospital, Rourkela, allowing imaging comparisons between different age groups within the same cohort. We first analyzed brain changes in CM survivors and observed two distinct patterns of cerebral swelling in both adult and pediatric patients, often developing simultaneously in distinct parts of the brain. The occurrence of posterior vasogenic edema caused by an impairment of the blood–brain barrier that reversed upon treatment was indicative of posterior reversible encephalopathy syndrome, which we demonstrated for the first time in the context of CM , a group first recruited to serve as control. Indeed, two earlier studies reported that mild to moderate brain swellingThe CSCMi MRI project benefited greatly from the long duration of the funding, which allowed nurturing of pivotal international collaborations, ultimately facilitating reproducibility and validation of imaging results. It has also led to the development of a range of research and clinical tools available to the malaria community, including the design of hypotheses-driven imaging protocols and quantitative methods to assess brain changes in falciparum malaria infection, and to the establishment of plasma biomarkers that strongly correlate with MRI findings and can therefore be used for diagnostic and prognostic approaches. These include lipocalin-2,P. vivax–associated morbidity and mortality has accumulated over the past decade,P. vivax in India, both at the acute and follow-up stages. Our first study in this area enrolled PCR-confirmed severe malaria patients at the Civil Hospital in Ahmedabad, Gujarat, followed over 12 months to compare morbidity and mortality between severe vivax and severe falciparum malaria.,Evidence of Our research and discoveries during the 12 years of the CSCMi highlight what can be achieved with continuous, long-term, stable funding and underscore the importance of undertaking basic and translational research on this important disease. A key factor has been the adaptability of the Center in the face of the changing epidemiology of malaria in India, making it responsive to new developments and questions as they emerge. For example, our studies on severe vivax malaria in Ahmedabad, Gujarat, were precipitated by a notable increase in number of cases in this area in 2016–2017, reported to us by clinicians at BJMC Civil Hospital in Gujarat. A central component of the CSCMi is interaction with malaria research, policy, and control counterparts in India to ensure that the research is relevant, significant, and strategic and that it provides opportunities for training, capacity building, and transfer of new technology. Our efforts and interactions in this area are described in the accompanying paper.


Plasmodium falciparum histidine-rich protein 2 (HRP2) or lactate dehydrogenase , were assessed against 159 samples collected from imported malaria cases, including 79 P. falciparum, 37 P. vivax, 22 P. ovale, and 21 P. malariae parasites. Samples had been previously characterised using microscopy and real-time PCR. The overall sensitivities for the Plasmodium test ranged from 69.8% (111/159) to 95% (151/159). There was no significant difference for the specific detection of P. falciparum . No significant difference was found between sensitivities to P. vivax by pan LDH or pvLDH (81.1% (30/37) to 94.6% (35/37) (p = 0.845)). Some of the RDTs missed most of P. ovale and P. malariae, with sensitivities for all RDTs ranging respectively from 4.5% (1/22) to 81.8% (18/22) and 14.3% (3/21) to 95.2% (20/21). Carestart Malaria Pf/Pan (pLDH) Ag G0121, a pLDH-based RDT (PfLDH and pLDH), showed the highest sensitivities to P. falciparum , P. vivax , P. ovale , and P. malariae  and meets the requirements for military deployments in malaria-endemic areas.Malaria is one of the most common tropical diseases encountered by members of the French military who are deployed in operations under constrained conditions in malaria-endemic areas. Blood smear microscopy—the gold standard for malaria diagnosis—is often not available in such settings, where the detection of malaria relies on rapid diagnostic tests (RDTs). Ten RDTs , based on the detection of the  Malaria remains a major problem for human health and in particular for the French military, which encountered more than 6000 cases between 2000 and 2015 [P. falciparum is predominant, but also in areas of the continent where the transmission of P. ovale is reported, such as Côte d’Ivoire, Gabon, and Senegal [P. vivax is predominant [P. falciparum, 29.0% of P. vivax, 7.2% of P. ovale, and 1.8% of P. malariae [Members of the French military are deployed in parts of Africa where  Senegal ,4,5. Mordominant . BetweenThe rapid diagnosis of malaria is essential due to the potential fast progression from uncomplicated malaria to severe malaria and death, especially in non-immune patients without appropriate treatment (such as the use of ACTs) ,8. In maPlasmodium falciparum-specific proteins and target either the P. falciparum histidine-rich protein 2 (HRP2) or the P. falciparum lactate dehydrogenase (PfLDH). RDTs can detect other pan-malaria (genus) antigens, such as pLDH or pALD . Some kits can detect the specific protein P. vivax lactate dehydrogenase (PvLDH). The users of the RDT and their institution must select the most efficient kit according to their needs . Moreover, the availability of a complete kit is essential. In each bag, there is an RDT, a solvent vial, an alcohol pad, and a needle. This helps to avoid the loss of the solvent vial or other essential elements, which are often sold in a single container for a whole box of 20–25 RDTs.Malaria rapid diagnostic tests (RDTs), based on immunochromatographic techniques, are complementary techniques used in laboratory settings in mainland France. RDTs are fast (results within 15–30 min of migration), and relatively easy to use and interpret . Howeverplasmodium in an endemic area within a constrained environment. The thin blood smear, the gold standard method, and real time PCR—the most sensitive and specific method—were used in parallel to check the validity of the results rendered by the RDTs. We hope this study will change the policy on the use of RDTs in the French military, in favour of a more sensitive ‘lab-in-a- kit’RDT package.The aim of our study was to compare the sensitivity of different RDT kits referenced in the World Health Organization (WHO) RDT testing report  and markP. falciparum (parasitaemia ranging from 0.001% to 21%), 37 P. vivax (parasitaemia ranging from 0.001% to 0.6%), 22 P. ovale (parasitaemia ranging from 0.001% to 0.6%), and 21 P. malariae (parasitaemia ranging from 0.001% to 0.4%).The isolates were collected from patients hospitalised in several civilian and military hospitals belonging to the French National Reference Centre for Imported Malaria network  between September 2018 and February 2019. The patients presented with imported malaria from a malaria-endemic country and their samples were sent to the French National Reference Centre for Malaria (CNR) . Whole blood was received in EDTA Vacutainer tubes after being transported from hospitals in the CNR. The samples were stored and transported at 4 °C from the moment of sampling until we receive the samples. A total of 159 samples were evaluated including 79 ® kit. Parasitaemia was computed on a minimum of 10 microscopic fields.Parasite species and parasitaemia assessments were carried out by certified operators on thin blood smears that were stained with eosin and methylene blue using an RAL® DNA Mini kit according to the manufacturer’s recommendations .DNA extraction for each sample was performed using the QIAampP. falciparum (NCBI:AJ413249), enoyl-acyl carrier protein reductase of P. vivax(NCBI:AY423071), circumsporozoite (NCBI:S69014), ookinete surface protein 25 (NCBI:AB074976) of P. ovale were amplified with Roche LightCyclerTaqMan master .Real time PCR was performed using the LightCycler 2.0 , as previously described . BrieflyP. falciparum but also P. vivax parasites [P. vivax in French Guiana where P. vivax is predominant. The first nine RDT kits approved and marketed in France were selected from the list of WHO validated tests. Among these nine RDTs, three were selected for their specificity to detect P. vivax parasites. The main criteria for selection were market availability and the ‘lab-in-a-kit’format. The RDT kit most commonly used by the French army health service at the time of the study , SD BiolineAlere Abbott) was added to the nine RDTs. The 10RDTs are presented in RDTs were selected according to the last WHO report on the performance of malaria RDTs on arasites . Membershttps://www.real-statistics.com/, accessed on 14 September 2022).The statistics were collated using an Excel spreadsheet and Charles Zaiontz’s Real Statistics add-on . The RDTs with the highest sensitivity to Plasmodium spp. (Carestart G0121) detected significantly more parasites than RDTs with a sensitivity below 86.8% .The sensitivities for all species of P. vivax by pan LDH or pv-specific LDH, which ranged from 81.1% to 94.6% .No significant difference was found between sensitivities to P. ovale parasites, with sensitivities for all RDTs ranging from 4.5% to 81.8% .Some of the RDTs  missed most of the P. malariae ranged from 14.3% to 95.2% . The RDTs which missed the P. malariae parasites were the same, with low sensitivities to P. ovale.The sensitivities for detection of P. falciparum . There was no cross detection between parasite species and the 10 different RDTs. However, on Palutop 4+, there were 15 out of 79 P. falciparum isolates that were positive on the “P. vivax LDH” band of the RDT (parasitaemia ranging from 0.25% to 12.7%). This has already been observed in the past, when Palutop 4+ was used in French Guyana, when sometimes when there is high parasitaemia or high levels of circulating HRP2, the P. falciparum HRP2 band is saturated and the P. vivax LDH turns positive. The two other RDTs with a P. vivax LDH band showed no cross reaction (Carestart and Humasis).There was no significant difference for the specific detection of It is vital for military personnel who are deployed with limited resources often without health personnel or reliable diagnostic means to be able to quickly detect the presence of malaria parasites. When there is no health facility nearby, ACT treatment must be administered directly in the field. In these conditions, clinical symptoms combined with the use of an RDT are the only clues the operator will have to diagnosis malaria. More than 200 malaria RDTs are currently commercially available worldwide and over 80 kits fulfil the criteria required by the WHO  . SeveralP. falciparum parasites with sensitivities ranging from 96.2% to 98.7%, while no differences were found in terms of specificity. Carestart Malaria Pf/Pan (pLDH) Ag (G0121) was the only one of the 10 RDTs which relied on specific pfLDH for the detection of P. falciparum. The nine other RDTs relied on HRP2 detection. The majority of the RDTs are designed to identify HRP2. Only 11/89 tests, based on the detection of pfLDH or both pfLDH-HRP2, met the WHO requirements for RDT procurement [P. falciparum [P. falciparum parasites lacking the pfhrp2 gene [pfhrp2-deleted mutants associated with false-negative results with HRP2-based RDTs have been reported in various countries  [The 10RDTs were efficient in the detection of curement . Previoucurement ,16,17. Tlciparum ,19 than rp2 gene , Caresta Rwanda) ,21,22,23 Rwanda) . The detPlasmodium spp which was under 90%  Ag G0121). These RDTs missed many P. ovale (4.5% to 59.1%) and P. malariae (14.3% to 71.4%). Some RDTs, such as Palutop 4+ Optima and SD Bioline Ag Pf/Pan 05FK63, were already known for their poor efficiency at detecting P. ovale and P. malariae [P. ovale (59.1% to 81.8%) and P. malariae (71.4% to 95.2%) than the other RDTs . Carestart Malaria Pf/Pan (pLDH) Ag G0121 was the most efficient at detecting P. ovale (81.8%) and P. malariae (95.2%).Five RDTs  showed global sensitivity to malariae . The fivP. vivax between the 10RDTs . Three RDTs, Malaria Pf/Pv Antigen Test AMFV-7025, Carestart Malaria Pf/Pv (HRP2/pLDH) Ag combo G0161 and Palutop 4+ Optima, specifically identified P. vivax (one specific band on RDTs) due to the detection of PvLDH (sensitivities from 83.5% to 91.9%) but were not more efficient at detecting P. vivax parasites than pLDH-based RDTs. The only advantage of these three RDTs would be that they could be used to detect P. falciparum-vivax mixed infections in areas where P. vivax was predominant and where there was a very low transmission of P. ovale and P. malariae parasites, such as in French Guiana and the Republic of Djibouti [P. vivax, only P. falciparum will be identified (one band-HRP2 or -PfLDH and one band-pLDH). If the RDT misses the P. vivax parasite, the patient will still be treated for uncomplicated falciparum malaria. This treatment includes artenimol-piperaquine or artemether-lumefantrine, which are the first-line curative treatments which are recommended in France for uncomplicated imported P. falciparum and non-falciparum malaria [P. vivax using primaquine is prescribed during a resurgence in the absence of G6PD deficiency, when only the P. vivax parasites are detected.There was no significant difference in the detection of Djibouti ,28. In t malaria . The samThe main limitation of this study was the low number of samples, although 80 non-falciparum samples were tested. There was no issue with sample preservation due to the short timescale within which the samples were sent to the CNR and the fact that the transport was maintained at 4 °C, as controlled by temperature tracker in each package.P. ovale and P. malariae and would therefore not be appropriate for either malaria diagnosis in the French armed forces or for cases of imported malaria. Carestart Malaria Pf/Pan (pLDH) Ag G0121, a non HRP2-based RDT, showed the highest sensitivities to P. falciparum (98.7%), P. vivax (94.6%), P. ovale (81.8%), and P. malariae (95.2%). This RDT meets the requirements of the French armed forces with regards to their military deployments in malaria-endemic areas: the time-to-results is rapid, it does not require extensive training or specific equipment, it is efficient for all Plasmodium parasites, and it can be used worldwide, even in a context of pfhrp2 deletions. This RDT also meets all the criteria for the diagnosis of imported malaria in non-malaria-endemic countries. As some of those RDTs meet the requirements of the French military forces in their deployments, they also could be useful for other applications linked with malaria diagnosis, such as in low-income countries where RDTs are used as a major diagnostic tool.This study showed that some RDTs missed the detection of


Low-density malaria infections (LDMI) are defined as infections that are missed by the rapid diagnostic test (RDT) and/or microscopy which can lead to continued transmission and poses a challenge in malaria elimination efforts. This study was conducted to investigate the prevalence of LDMI in febrile cases using species-specific nested Polymerase Chain Reaction (PCR) tests in the Malaria Elimination Demonstration Project, where routine diagnosis was conducted using RDT.Plasmodium species.Every 10th fever case from a cross-sectional community based fever surveillance was tested with RDT, microscopy and nested PCR. Parasite DNA was isolated from the filter paper using Chelex based method. Molecular diagnosis by nested PCR was performed targeting 18SrRNA gene for The prevalence of malaria was 2.50% (436/17405) diagnosed by PCR, 1.13% (196/17405) by RDT, and 0.68%  by microscopy. Amongst 17,405 febrile samples, the prevalence of LDMI was 1.51% (263/17405) (95% CI 1.33–1.70), which were missed by conventional methods. Logistic regression analysis revealed that illness during summer season [OR = 1.90 (p < 0.05)] and cases screened within three days of febrile illness [OR = 5.27 (p < 0.001)] were the statistically significant predictors of LDMI.The prevalence of malaria among febrile cases using PCR was 2.50% (436/17405) as compared to 1.13% (196/17405) by RDT. Higher number of the LDMI cases were found in subjects with ≤ 3 days mean duration of reported fever, which was statistically significant (p < 0.001). This observation suggests that an early detection of malaria with a more sensitive diagnostic method or repeat testing of the all negative cases may be useful for curtailing malaria transmission. Therefore, malaria elimination programme would benefit from using more sensitive and specific diagnostic methods, such as PCR. Malaria in India is heterogeneous and complex disease because of its population, topography, epidemiology, and diverse climatic conditions. In 2020, globally, approximately 241 million malaria cases were detected. In the South East Asia region of the World Health Organization (WHO-SEARO), malaria case incidence has been reduced by 83% in the past two decades; from about 18 cases per 1000 population at risk in 2000 to about 3 cases in 2020  [Genomic DNA was isolated from the dried blood spots (collected during Jan 2018 to Dec 2019) using Chelex method [Chelex-100 Sodium form (50–100 Mesh) Himedia Laboratories]. In brief, 3 punches of filter paper were cut and soaked in 1 ml of 0.5% saponin in phosphate buffered saline (PBS), vortexed for 20–30 s and incubated overnight at 4 °C. After incubation tubes were centrifuged at 3000 rpm for 60 s, fluid was aspirated and the filter paper was crushed in 100 µl of Mili Q water. After this step 50 µl of a stock solution of 20% Chelex-100 was added to these 1.5 ml tubes and heated at 95–99 °C in a heating block for 12 min and vortexed for every 2–3 min. The tube was centrifuged at 8000 rpm for 5 min the supernatant recovered, further the tube was centrifuged under the same condition for 10 min. the supernatant was collected in a new tube and stored at − 20 °C for further process . The pret. Ltsd] . The detThe demographic  variables, clinical symptoms related to malaria, results of RDT, microscopy, and PCR were entered in Microsoft Excel 2007 worksheet and numerically coded data was exported in R 4.1.2 for Windows  for statistical analysis. Sensitivity, specificity, positive predictive value (PPV), negative predictive value (NPV) with a 95% confidence interval of Microscopy and RDT against PCR as gold standard were calculated. The sensitivity was calculated as the number of true positives/ , the specificity as the number of true negatives/ , the PPV as the number of true positives/  and the NPV as the number of true negatives/. Logistic regression analysis is used to examine the association of independent variable(s) with LDMI.P. falciparum, 52 P. vivax and 8 mixed infection of P. falciparum + P. vivax) were found positive by RDT. Amongst the RDT negative cases , 17 cases (0.1%) and 280 cases (1.63%) were found positive by microscopy and PCR, respectively. Further, out of the 136 P. falciparum positive cases by RDT, 70 (51.47%) cases tested positive by microscopy and 109 (80.14%) tested positive by PCR.A total of 195,279 febrile cases were screened using RDT, out of which 17,405 samples were screened by light microscopy and PCR. The prevalence of malaria was 2.50% (436/17405) diagnosed by PCR, 1.13%  by RDT and 0.68%  by microscopy. Out of these 17,405 febrile cases, 196 cases  and 35 (67.30%) were also confirmed as P. vivax by microscopy and PCR, respectively. However, one case was diagnosed as P. falciparum and 2 were found to be mixture of P. falciparum and P. vivax using PCR , and two negative cases. None of these were found positive by microscopy . Out of the 476 malaria cases, 101 (21.22%) cases were found positive by all three diagnostic methods. Seventeen of 476 cases were positive by microscopy and PCR, but negative by RDT. There were 55 cases positive by RDT and PCR but negative by microscopy  microscopically positive individuals. Whereas age group wise prevalence showed that the percentage of gametocytes was highest 67% (4/6) among 8–14 years of age group Tables  and 3.P. falciparum, the sensitivity and specificity of RDT and microscopy against PCR was 32.5%, 99.8%, and 25.1%, 100% respectively. However, the sensitivity and specificity of RDT and microscopy for P. vivax was 44.9%, 99.9%, and 38.5%, 100%, respectively ] and screened within three days of febrile illness [OR = 5.27 ] were the statistically significant predictors of LDMI. The multivariate logistic regression analysis showed that the cases screened within three days of febrile illness was a highly significant predictor of LDMI , with the controlled effect of all other independent factors such as endemicity, season, and age  of he blood , 28.Similarly, microscopy can have limitations, such as only properly trained microscopists can detect low density parasitaemia and mixed-species infections , 29. TheThe present study was designed to determine the prevalence of LDMI amongst the fever cases in district Mandla of Madhya Pradesh, India using nested PCR tests. The study revealed that the malaria prevalence was 2.73% 476/17405) in febrile cases by using all three diagnostic methods. Similar low levels of malaria positivity in fever survey was reported in a previous study where RDT was used as the sole diagnostic tool under the T4 strategy [ in febriPfhrp2/3 gene deletion resulting in a false negative result. Others studies have reported Pfhrp2 gene deletion in the entire country [Currently, RDT is considered as the principal diagnostic tool for community-based diagnosis of malaria in India. In the present study, only 1.13%  malaria positivity was recorded by RDT, whereas 2.50% (436/17405) positivity was recorded by PCR. There were 17 cases diagnosed positive by microscopy and PCR, but negative by RDT. This could be due to  country , 30.Out of 476 malaria positive cases, 40 (8.40%) were found positive by RDT and missed by both PCR and microscopy, which could be due to circulation of Pfhrp2 in the blood from past infections. It has been previously reported by Kyabayinze et al.  that HRPOut of the total prevalence of malaria (2.73%) amongst the febrile cases, more than half (1.51%) were LDMI that were only detected by PCR. Similar study conducted in two districts of Chhattisgarh between 2007 and 2008 showed that 66.3% of peripheral and 64.4% of placental infections were submicroscopic . AnotherPlasmodium infections were reported amongst the asymptomatic individuals by qPCR with 37% submicroscopic malaria [In another high endemic area of Kandhamal district in Odisha, 18%  malaria . The imp malaria  and Ethi malaria . ConsideWe observed a high prevalence of LDMI during the dry season (April–May) which is consistent with the findings of Dielmo and Ndiop in Senegal . This stIn the present study, it was observed that LDMI was likely to be associated with early febrile illness within 3 days. Early-stage malaria infection can be missed by microscopy and RDT as the sensitivity of microscopy and RDT is considerably lower (in the range of 40–100 parasites per µl of blood for microscopy and approximately 100 parasites per µl of blood in approximately 5 µl of whole blood for RDTs) , 39. A sOther studies have also reported an association of sub-microscopic malaria with fever and non-febrile illness , 42. In Plasmodium infections [As compared to microscopy and rapid diagnostic tests, the PCR methods are used in malaria surveillance as well as epidemiological research . The usefections . From a This study has revealed that RDT and microscopy miss a significant number of low-density malaria infections. Though the approved and available RDT/microscopy-based diagnosis will stay as the diagnostic test of choice in case management of malaria, the results of this study suggests that PCR-based diagnostic tests should be used in malaria elimination programmes to determine true case-load of malaria among the febrile cases from high transmission areas. The present WHO guidance is to use RDTs and microscopy for malaria diagnosis, however, they also recommend use of PCR on a research-mode to develop a body of evidence for its use in surveillance. This study provides useful data for malaria elimination programmes to consider.


Plasmodium falciparum and vivax. Using geostatistical methods and remote-sensing data we assessed the environmental and spatial associations with exposure, and created predictive maps of exposure within the study sites. We additionally linked the active surveillance PCR and serology data with passively collected surveillance data from health facility records. We aimed to highlight the added information which can be gained from serology as a tool in active surveillance surveys in low-transmission settings, and to identify priority areas for national surveillance programmes where malaria risk is higher. We also discuss the issues faced when linking malaria data from multiple sources using multiple diagnostic endpoints.The epidemiology of malaria changes as prevalence falls in low-transmission settings, with remaining infections becoming more difficult to detect and diagnose. At this stage active surveillance is critical to detect residual hotspots of transmission. However, diagnostic tools used in active surveillance generally only detect concurrent infections, and surveys may benefit from sensitive tools such as serological assays. Serology can be used to interrogate and characterize individuals' previous exposure to malaria over longer durations, providing information essential to the detection of remaining foci of infection. We ran blood samples collected from a 2016 population-based survey in the low-transmission setting of northern Lao PDR on a multiplexed bead assay to characterize historic and recent exposures to  P. vivax cases making up the majority of malaria burden  data collected from health facilities in the same districts  of northern Lao PDR , which arovinces . It is arovinces , 3. The The passive surveillance data were gathered according to Rerolle et al. . BrieflyThe survey data were collected according to Lover et al. . BrieflyP.falciparum and P.vivax antigens ] diluted 1:200 as described previously , P. vivax positive control  and a curve of pooled hyperimmune P.falciparum sera  were included in singlicate on each test plate to assess interplate variability. 96 malaria-naïve sera from the UK  were assayed at 1:100 using the same method.The chemical coupling of 17 antigens  to MagPlu et al. . 3 mm pueviously . BackgroPlasmodium DNA using a qualitative real-time PCR assay which targeted Plasmodium cytochrome b gene  denotes seroprevalence at locations xi, i = 1, …, n, the number of positive households Yi out of Ni people sampled follows a binomial distribution:Geostatistical models of household seroprevalence for each species exposure were fit separately for the 1,402 households in the active surveillance. The models were fit within a Bayesian framework where 0 denotes the intercept, d(xi)′β denotes a vector of location specific covariate effects (within active survey district boundaries) and wi represents the spatial effect. The spatial effects were modeled as a Matérn covariance function using the stochastic partial differential equation (SPDE) approach in Integrated Nested Laplace Approximation (R-INLA)  . The inthreshold . The uppTo evaluate health facility catchment-level seroprevalences and PCR prevalences, we estimated catchment areas for all health facilities from an official Lao PDR Ministry of Health list  of 190 hApprovals for the field surveys (PI: Adam Bennett) were obtained from UCSF  and the Lao National Ethics Committee for Health Research, Lao Ministry of Health . Both approvals included provisions for future analysis of serological markers of malaria exposures.P. vivax cases and 36 P. falciparum cases were confirmed by RDT. 23 P. vivax cases and 12 P. falciparum cases were confirmed by microscopy.The results from the retrospective survey of 2016 passive surveillance records from the study regions are presented in P. falciparum and P. vivax are as follows: PfMSP119 541.26 (±1334.88); PfAMA1 723.33 (±1785.43); Etramp5.Ag1 99.31 (±183.71); PvMSP119 169.272 (±181.789); PvAMA1 483.1 (±1772.88); PvEBPII 371.64 (±860.05). The range of individuals' MFI values by positivity and age are presented in The mean MFI values for the antigens used to define historic and recent exposure to P. vivax and P. falciparum.P. vivax were higher (0.22 for historic and 0.07 for recent) than P. falciparum (0.03 for historic and 0.01 for recent). The age-stratified seroprevalences for each species exposure are shown in P. falciparum and recent exposure to P. vivax. These increases began at around 25 years of age for both exposures. Recent exposure to P. falciparum was very low across all age groups.At the survey-level seroprevalences for exposures to P. vivax (n = 23) than P. falciparum (n = 8). Of the 23 positive P. vivax PCR cases in the active survey, seven were classified as positive for recent exposure to P. vivax (30%). Of the eight positive P. falciparum PCR cases in the active survey, one was classified as positive for recent exposure to P. falciparum (12.5%). The central districts of Khua and Nambak recorded the highest number of positive PCR P. vivax cases. P. falciparum PCR case numbers were similar across the three study areas.The PCR survey also found higher case numbers of P. vivax and P. falciparum exposure. The environmental and spatial covariates included in the final geostatistical models are listed in P. vivax and P. falciparum and RDT and microscopy cases per capita are presented in The estimation of catchment size resulted in 190 catchments across Lao PDR . Due to P. vivax and P. falciparum were more widespread in northern Lao PDR, with recent exposures being more focally distributed, as is expected in an elimination setting. Additionally, we showed how retrospectively collected passive surveillance data can be linked to active surveillance data which were not collected in alignment.This study has demonstrated the use of integrating serology into active surveillance projects to provide additional information on historic and recent exposures to malaria. We have shown how geostatistical modeling with remote sensing-derived environmental variables can be used to predict and characterize the distribution of malaria exposures, and how these can be used to highlight priority areas for added data collection or targeted interventions. We found that historical exposures to Plasmodium infections, with higher numbers of P. vivax  and lower numbers of P. falciparum . The seroprevalence rates estimated in this study for recent exposure to P. vivax and P. falciparum were higher than these PCR rates but followed similar trends. The higher numbers of P. vivax cases compared to P. falciparum detected by Lover et al. . Here, and in Lao PDR, malaria transmission is heterogenous, and transmission is more intense in forested areas , alongside measures of uncertainty . This study was funded by the Bill and Melinda Gates Foundation (OPP# 1177272).The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


Malaria is a serious problem in children because the immune system is lessdeveloped, thus, causing more severe symptoms. This study aimed to identifyfactors associated with malaria in indigenous and non-indigenous patientsaged under 15 years in Amazonas, Brazil, from 2007 to 2018.  An epidemiological, quantitative, cross-sectional study was conducted. Casesincluded patients aged under 15 years, using data from health systemnotifications between 2007 and 2018 in the state of Amazonas, Brazil. Thevariables included clinical-epidemiological, laboratory findings, andmonitoring of cases. The outcome was ethnicity: indigenous, non-indigenous,and entries for which no ethnicity data were provided. A multivariablelogistic regression model was used to compare the indigenous andnon-indigenous populations. Plasmodium falciparum infections were more frequent,and timeliness of treatment, i.e., the interval between the onset ofsymptoms and time of treatment was within 48 hours.  Among malaria cases in patients aged under 15 years, there was a greaterchance of being indigenous and having the following associated factors:female sex, children aged 0-4 years, passive case surveillance, a high loadof parasitemia and the lack of data regarding the level of parasitemia, The factors associated with malaria are more frequent in indigenouspopulations and highlight differences according to ethnicity, suggestingthat the severity of the disease is attributable to the increased number ofmalarial infections within this population. As a result, malaria has agreater impact on the health of indigenous people. Plasmodium and is transmitted to humans by female mosquitoes ofthe genus Anopheles. Two species of parasites are important forpublic health in Brazil: Plasmodium vivax, which sometimes causesmild symptoms, and P. falciparum, which has a greater potential formalaria-related deathsMalaria is an infectious disease caused by protozoa belonging to the genusAnopheles darlingi vectorIn Brazil, the epidemiological situation of malaria is most severe in the Amazonregion, mainly when analyzed in incident cases, as almost all cases are registeredin this part of the countryAlthough the disease affects all age groups, it is a greater threat to childrenconsidering the following factors: the potential of the disease to deterioratehealth, its difficult assessment as most clinical symptoms are non-specific, andmost cases occur in places where routine malaria tests are not readilyavailable-Malaria does not endanger a person’s health during the active phase of the disease.However, the consequences of malaria interfere with the quality of life andproductivity of the population and, in children, can jeopardize development andlearningStudies have pointed to the severity and poor care of indigenous children andadolescents, who were the main victims of the malaria outbreak that hit the UpperNegro River in 2018. It affects 60% of this population groupThis is a cross-sectional epidemiological study that used secondary data from theInformation System for Epidemiological Surveillance of Malaria .The retrieved data included children and adolescents aged under 15 years withmalaria between 2007 and 2018, and when the state of Amazonas was reported as aplace of residence and probable infection setting.-This age group was selected because the available data were stratified by agegroup and not by age. Nevertheless, we did not analyze mortality, and weincluded children and adolescents aged under 15 years, regardless of whetherthis is mixed or not using only children, as children have been used in otherresearchAll malaria cases involving patients aged under 15 years who reported to theSIVEP-Malaria between 2007 and 2018 were included in the study population.Sampling criteria were not considered, and all records were included. Regardingthe inclusion criteria, all Amazonas residents with the same state as a probablesite of infection were considered. Therefore, imported cases were excluded fromthe analysis.It is important to note that since these are secondary data obtained without theidentification of the persons, records only reflect episodes of malaria and notthe patient itself. In view of this, there may be people with more than oneepisode of malaria within the study period and thus are included in the study.In addition, if the registers were “cure check slides”, they were excluded asthey had already been reported as a new case and had a previous entry in thedatabase.® 13.0 software (StataCorp). These data were reviewed toseparate indigenous and non-indigenous, and entries for which no ethnicityinformation was given. The samples were then filtered again to identify thecases for which Amazonas was given a place of residence and probable place ofinfection.The SIVEP-Malaria data were made available by the Amazonas Health SurveillanceFoundation (FVS-AM), obtained in an Excel spreadsheet format, and analyzed usingStata-For the definition of “indigenous,” the criterion of another study on indigenouspeople in Amazonas, Brazil, was adopted due to the high number of entries withmissing data in the “ethnicity” field, which represented 51.4% of thenotifications in the study periodThe variables used in the analyses were grouped as following:P.falciparum ; 2- P.vivax; and 3- mixed , and others,including P. malariae and P.ovale;Clinical-epidemiological and laboratory findings were as follows: sex; age group ; type ofsurveillance slide (active or passive case surveillance);parasitemia level ; test results , the interval between first symptoms and treatment(days), and treatment opportunity (relationship between date ofdiagnosis and start of treatment), which was considered early whenit was less than 48 hours. For the descriptive phase, data were analyzed according to frequency anddistribution and presented as percentages for categorical variables. In the analytical phase, we performed a bivariable analysis using Pearson’schi-square test. The effect size was evaluated using Cramer’s V coefficient,including indigenous, non-indigenous, and missing data on ethnicity (missingdata) as a comparison factor. We chose to display the missing data to present anoverview of the situation and test in the multivariable analysis to checkwhether the lack of data would alter the association of malaria in indigenouspatients aged under 15 years. We calculated the standardized residuals of the chi-square test among thecategorical variables, which allowed us to identify the characteristic patternsin each category of a variable according to the excess or lack of occurrence ofthe combination of each category of the other variables. This allowed us tounderstand the significance of the associations, especially within the category.The residuals of the chi-square test with positive values greater than 1.96corresponded to a level of significance of 5% and 95% confidence interval(CI)P-value was used for inclusion in the model from the bivariableanalysis. Variables with statistical significance (P < 0.05)were retained in the final model, and the results were reported using oddsratios (OR) together with their 95% CI. Model fit was evaluated using theHosmer-Lemeshow testFor multivariable analysis, logistic regression adjusted in stepwise forward wasused, and the outcomes were indigenous and non-indigenous. In the modelingprocess, the < 0.10 criterion of the significance of theAs the data used had already been registered in the SIVEP-Malaria system and theethnicity classification was completed at the time of notification of the case,there was no need for consent because the authors did not contact the patients,and the data were made available without identification. The Research EthicsCommittee approved the project at the Federal University of Amazonas underprotocol number 2.302.738.The results are presented in tables containing the characteristics of the patients, clinicaP-values (< 0.01). Regarding the indigenous age group, therewas a higher frequency of malaria in children aged under five years, whoseproportion decreased as the age group increased, in contrast to what occurs innon-indigenous and in cases of missing data for ethnicity  in the standardizedresiduals of the chi-square test: (a) in non-indigenous - active case surveillance,lower parasitic load (up to one cross), and P. vivax infection; (b)in cases lacking information on ethnicity (missing data) - active case surveillance,two or more crosses of parasitic load, and disease caused by some stages form ofP. falciparum parasite; (c) and in indigenous - passive casesurveillance, three or more crosses of parasitic load or missing data in the crossesnumber field, and disease caused by some stages form of P.falciparum parasite, P. vivax, or other parasites.P-values (< 0.001), as shown in The association between the number of days between the date of onset of symptoms anddiagnosis and ethnicity, as well as the time between diagnosis and the start oftreatment and ethnicity, were found to be small in terms of effect size evaluatedusing Cramer's V test , with statistically significantRegarding the number of days between the date of onset of symptoms and diagnosis, theexcess occurrence was 0 days and more than 7 days in the indigenous populations. Thenon-indigenous populations showed excess occurrence for 1, 2, 3, and > 7 days.The missing data on ethnicity presented significant residuals of the chi-square testfor either 1 to 7 days, indicating no distinction for early diagnosis in thiscategory. Regarding the time between diagnosis and the start of treatment, theresult was similar to the days between the first symptoms and treatment, except forcases in which there was no data on ethnicity, with significance in the residuals ofthe chi-square test for 3 days or more .The results of the multivariable logistic regression analysis, which estimated thefactors associated with malaria cases in indigenous and non-indigenous patients agedunder 15 years, with the respective values of crude and adjusted ORs, are presentedin All variables tested in the bivariate logistic analysis were statisticallysignificant for inclusion in multivariate analysis. In the final model, we highlightthe factors associated with malaria in indigenous compared with those non-indigenouschildren and adolescents, which presented the greatest magnitude: female sex ; age group younger than 1 year ,with a reduction of the OR as age increases, maintaining statistical significance;passive case surveillance ; a high load of parasitemia and the lack of information on parasitic load field; mixed parasitic infections  ; timeliness of treatment, i.e., the start of treatment within 48 hfrom the date of the first symptoms  .The factors associated with malaria among reported cases of children and adolescentswarrant attention and proper care in this population group. The differences observedbetween indigenous and non-indigenous patients aged under 15 years indicate that thedisease affects ethnic groups in different ways.The association between malaria in patients aged under 15 years and female sex is anenigma; such that one would not expect sex to have a great significance despite thepredominance of male sex in the study population, nor a difference in theassociation between non-indigenous and that of which is already known in adultsDetecting a stronger association of malaria in the younger age group is an indicatorof the severity of the problem in this population. Children aged under 1 year havean immature immune system, making them more vulnerable to infectious agentsMalaria is widely distributed in non-indigenous people aged 10 to 14 years and may berelated to intra-and peridomicile transmission if we consider housing conditions andproximity to forest areas, vector density, or basic sanitationIndigenous people have specific health policies that the National Health Service(Sistema Único de Saúde - SUS) should adapt to and consider when creatingorganizational models that aim to promote health and prevent the spread of malariafor this groupIn indigenous populations, among the reported malaria cases, passive casesurveillance is more frequent for cases of infection in children aged under fiveyears, possibly due to mothers taking their children to healthcare facilities assoon as their first symptoms appear. In contrast, in non-indigenous populations,active case surveillance with high occurrence demonstrates the efficiency ofinvestments and efforts established to ensure an increased network of malarialaboratories throughout the Amazonas state, with an increment of 72% in 10yearsPlasmodium; 29% ofinfections diagnosed by thick blood smear are that of P. vivax,when analyzed by polymerase chain reaction (PCR), were identified as mixedinfectionsOne of the most commonly used techniques for the laboratory diagnosis of malaria is athick blood smear that seeks specific confirmation of the disease. This technique isimportant because it allows visualization of parasites, species identification, andstages of development and quantification, which are essential data for clinicalevaluationPlasmodium species present, P. vivaxwas shown to be an infectious agent prevalent in non-indigenous populations, whichis similar to another studyP. vivax establishes itself under highertemperature conditionsP. falciparum, the most common parasite wasP. vivax.Regarding the P. falciparumcases, even though the total number of cases remained relatively high. Hyperendemictransmission is thought to be affected by extensive anthropogenic environmentalchanges in conjunction with the way of indigenous life in the region, which arelikely to have extended the habitats and increased the density of vectors, renderingvector control tools insufficientThe efforts of the program soon led to a reduction in P. falciparum and mixed malaria are predominantin indigenous people, as they are more severe in these cases. However, as symptomsexacerbate, this contributes to the immediate search for a diagnosis since passivemalaria case surveillance has the largest percentage among indigenous people. Inaddition, indigenous people aged under 15 years have diverse parasitic forms ofinfection, with a prevalence of mixed infections .It is worth noting that cases of P. falciparum infection is extremelyalarming, especially if people live in isolated areas, where traditional healthsystem practices are largely unavailable, and remains a considerable challenge forcase detection and proper follow-up. It is important to note that the literature hasrevealed the possibility of vulnerability arising from the Duffy-negative (Fy-)blood group, leading to the predominance of P. falciparum. However,this aspect was not investigated in this studyThe high proportion of The timeliness of treatment of malaria among indigenous people in less than 48 hoursmeets the recommendations set by the Brazilian Ministry of Health and is animportant factor for the control of the disease; the earlier the treatment, thelower the possibility of spread by reducing the source of infection. It is also anindicator of the level of care provided because in the context studied, indigenouschildren and adolescents had both diagnosis and received treatment earlier comparedwith those non-indigenous. It may also be attributed to the prompt care they receiveas soon as the first symptoms appear and the immediate pursuit of health serviceswhere diagnosis and treatment are provided. It is also possible that it is relatedto the delivery of health services in indigenous areas, in which access isfacilitated due to close proximity to the area. For example, the person who usuallyperforms the thick blood smear examination is a professional living in the area oreven an indigenous professionalThe data presented by the SIVEP-Malaria system, like any secondary data, presentspotential biases that are out of the researchers’ control, such as the lack ofstandardization in data collection, which affects the quality of the records, thecoverage that can vary in time and space, and lack of information, as in the case ofthe variable ethnicity and parasitemia, both showing a high percentage of lack ofdata. In the first case, it was demonstrated that the classification method used inthis work, including the indigenous village as a place of probable infection,contributed to the increase in ethnicity data completeness. However, in the secondcase, the lack of quantitative information about parasitemia was used in theanalysis, thus, limiting the interpretation of the parasitic burden. For missingparasitic data, there is a possibility of both high and low parasite loads becauseboth make counting difficult and require greater microscopist skill, which may be anadditional vulnerability in indigenous areas. Although these limitations have notcompromised the interpretations of this study, additional analyses are necessary tounravel both the epidemiological situation of malaria involving indigenous peopleand the effectiveness of the actions focused on indigenous health aimed at theprevention and control of endemic malaria. The factors associated with malaria in indigenous children in Amazonas may indicatethat the disease is facilitated by their way of life and the organization of healthservices in that region. Understanding these factors contributes to the developmentof strategies to provide proper healthcare to populations who are often vulnerabledue to geographical, economic, or biological reasons.Plasmodium infection in these areas. The results suggest that actions appropriate to the peculiarity of this populationneed to be developed to reduce the incidence of malaria in children and adolescents,especially among those aged under 5 years; thus, this group may not experiencefurther harm, such as the likelihood of repeated infections when malaria is acquiredearly. Therefore, malaria control programs should be implemented to improve effortsto contain the disease in these groups. Furthermore, the results of this researchmay guide the delineation of more effective and assertive decisions by professionalsresponsible for reducing


Plasmodium knowlesi infections in Malaysia are a new threat to public health and to the national efforts on malaria elimination. In the Kapit division of Sarawak, Malaysian Borneo, two divergent P. knowlesi subpopulations (termed Cluster 1 and Cluster 2) infect humans and are associated with long-tailed macaque and pig-tailed macaque hosts, respectively. It has been suggested that forest-associated activities and environmental modifications trigger the increasing number of knowlesi malaria cases. Since there is a steady increase of P. knowlesi infections over the past decades in Sarawak, particularly in the Kapit division, we aimed to identify hotspots of knowlesi malaria cases and their association with forest activities at a geographical scale using the Geographic Information System (GIS) tool. A total of 1064 P. knowlesi infections from 2014 to 2019 in the Kapit and Song districts of the Kapit division were studied. Overall demographic data showed that males and those aged between 18 and 64 years old were the most frequently infected (64%), and 35% of infections involved farming activities. Thirty-nine percent of Cluster 1 infections were mainly related to farming surrounding residential areas while 40% of Cluster 2 infections were associated with activities in the deep forest. Average Nearest Neighbour (ANN) analysis showed that humans infected with both P. knowlesi subpopulations exhibited a clustering distribution pattern of infection. The Kernel Density Analysis (KDA) indicated that the hotspot of infections surrounding Kapit and Song towns were classified as high-risk areas for zoonotic malaria transmission. This study provides useful information for staff of the Sarawak State Vector-Borne Disease Control Programme in their efforts to control and prevent zoonotic malaria. Zoonotic malaria by the simian parasite Plasmodium knowlesi became prominent since the large focus of cases reported in Kapit division of Sarawak state, Malaysian Borneo almost two decades ago2. Knowlesi malaria have been reported in countries across Southeast Asia at low frequency, however, highest prevalence of clinical cases has mainly occurred in Malaysian Borneo3. According to the Ministry of Health Malaysia, the prevalence of indigenous malaria caused by human parasites P. vivax. P. falciparum, P. malariae and P. ovale has shown a remarkable decrease while knowlesi malaria cases have continuously shown an increasing trend with 509 annual cases reported in 2010, to between 1813 and 4124 cases from 2012 to 20204 . Malaysia is listed by the WHO as one of the countries that has substantially progressed in eliminating malaria by the year 20201. However, zoonotic malaria cases caused by P. knowlesi are excluded from the definition of malaria elimination by WHO, which focuses on only the human Plasmodium species6.Malaria, a mosquito-borne disease, is widely distributed in the tropical and subtropical regions, with more than 400,000 annual deaths reportedAnopheles mosquitoes. Nonetheless, certification of malaria-free status for Malaysia by the WHO could be postponed if hundreds cases of knowlesi malaria per year are continuously being reported7.The malaria-free status by WHO is confirmed if zero incidence of indigenous cases for at least three consecutive years, denoting full interruption of local malaria by P. knowlesi has been shown to be complex. To date, there are at least three genetically divergent parasites that can infect humans, based on the analyses of multi-locus microsatellites markers and whole-genome sequences derived from clinical samples across Malaysia11. Two divergent subpopulations of P. knowlesi have been identified sympatrically in human infections in the Kapit division12; one (termed Cluster 1 subpopulation) is associated with long-tailed macaques (Macaca fascicularis) and the other (Cluster 2) is linked to pig-tailed macaques (Macaca nemestrina). Additionally, an exclusive P. knowlesi subpopulation (Cluster 3) has also been described in Peninsular Malaysia, indicating allopatric divergence from Cluster 1 and 2 subpopulations of the Malaysian Borneo due to geographic separation by the South China Sea10.The transmission of P. knowlesi Cluster 1 subpopulation is consistently predominant across Malaysian Borneo, and in the Kapit division of Sarawak, Malaysian Borneo accounts for two thirds of all cases with no significant temporal changes over the past 18 years13.Further large-scale genotyping surveillance using simple PCR tools showed that the 14. Human activities at the forest or forest fringe are one of the main risk factors for P. knowlesi infections, as it requires the presence of both macaque hosts and forest-dwelling Anopheles mosquitoes16. Individuals engaged in agricultural activities, hunting, and logging contribute to almost all cases of P. knowlesi infections in both Sarawak and Sabah states of Malaysian Borneo17. With the increasing cases and existence of two sympatric subpopulations in Malaysian Borneo, it is important to determine the spatio-temporal patterns of these P. knowlesi subpopulations particularly in the Kapit division of Sarawak where high incidence occurs, as well as the association with environmental changes in order to understand the transmission dynamics of this zoonotic parasite. Therefore, this study aimed to identify hotspots of P. knowlesi infections in the Kapit division and to determine the association with risk activities at a geographical scale.It has been suggested that man-made activities and environmental modifications trigger the increasing cases of knowlesi malaria2 with 134,000 inhabitants, mostly (49%) residing in Kapit, followed by Belaga (33%) and Song (18%) districts18. According to the Kapit District Council, the topography of the Kapit division varies from lowland to mountainous landscapes, with approximately 80% covered with dense primary forests. The Rejang River and the main upper tributaries, which include Batang Baleh, Batang Katibas, Batang Balui, and Belaga river, flow throughout the division and there were approximately 534 longhouses altogether, most located along the rivers.This study was conducted in the Kapit division, Sarawak, located at the central part of Malaysian Borneo Fig. . Kapit dKapit Hospital is the only hospital in the Kapit division, so all patients diagnosed with malaria at government health clinics are referred to this hospital. Approximately 2 mL of venous blood was collected from each patient at Kapit Hospital from June 2018 to December 2019. Each sample was used to prepare blood spots on filter paper and thick and thin blood films, and the remaining blood was frozen. The baseline information, including age, sex, and parasite microscopic examinations were also recorded at the hospital laboratory as part of the routine diagnosis for malaria. All samples were then transferred to the Malaria Research Centre, Universiti Malaysia Sarawak, for further molecular analyses.Written informed consent was obtained from enrolled patients or parents or guardians for patients below 17 years of age. All procedures were performed in accordance with relevant guidelines outlined in the ethical clearance. Ethical clearance for this study was obtained from the Medical Ethics Committee of Universiti Malaysia Sarawak (UNIMAS/NC-21.02/03-02 Jld.2 (81)), and from the Medical Research and Ethics Committee of the Ministry of Health, Malaysia (NMRR-16-943-31224(IIR)).Plasmodium DNA was extracted from dried blood spots using InstaGene™ Matrix , and the identification of Plasmodium species  was conducted by nested PCR assays as described previously19. For infections positive with P. knowlesi, each sample was further genotyped using allele-specific PCR assays to identify the two subpopulation clusters13.P. knowlesi infections in the Kapit division from January 2014 to May 2018 obtained from previous studies ). Data of each infection obtained were of a 6-year period from January 2014 to December 2019, which includes gender, ethnicity, occupation, travel history, activities prior to infection, and geographical coordinates. The database was reorganised accordingly in Microsoft Excel by excluding non-P. knowlesi infections and other irrelevant information. The map of the Kapit division was obtained from DIVA-GIS free spatial data depository (http://www.diva-gis.org). The coordinate system was projected into Timbalai 1948 RSO Borneo Meters in order to synchronise the digital data structure used in ArcGIS.Demographic data of patients with 20, which measures the economic workforce by rationing the number of dependents (non-working age) into the working-age population. Age < 15 and > 64 years old were considered as economically dependent and the age between 15 and 64 was reflected as an active working-age group. In Malaysia, the compulsory schooling age ends at 17 years old, so the working-age was adjusted at 18 years old.Patients were categorised into four age structures based on the dependency ratio in MalaysiaPatients were further categorised into several groups based on their activities two weeks prior to admission to the hospital for malaria. These activities were then categorised according to the distance radius from the longhouses and types of activities. Additionally, occupations were also categorised into six major groups, depending on the distance from the forests and working sites  was used to measure the Nearest Neighbour Ratio (R) based on the observed average distance between the nearest neighbouring infections21  interpolation technique was used to determine the hotspot areas of P. knowlesi subpopulations13, normalisation was applied to adjust the default density values of the KDE analysis. This function was set to avoid biased results when determining hotspot areas for the two P. knowlesi subpopulations. The density values for each hotspot result were set as default, calculated according to Silverman's Rule of Thumb algorithm in the software programme. To alter the density values, the P. knowlesi subpopulation hotspot values were customised according to the total malaria cases density values in that particular year.Due to the disproportion frequencies between Cluster 1 and Cluster 2 P. knowlesi infections, and 13 (3.0%) had double infections of P. knowlesi mixed with other human Plasmodium species  patients identified with non-P. knowlesi infections had all recently returned to Kapit after working in timber camps in malaria-endemic countries in Africa, South America and the Western Pacific islands.We obtained 436 blood samples from malaria patients admitted at Kapit Hospital from June 2018 to December 2019 with parasitaemia ranging from 20 to 384,000 parasites/μl blood . By PCR assays, 304 (69.7%) were positive for single P. knowlesi single or mixed infections, 74.1% (n = 235) were genotyped as Cluster 1 subpopulation and 15.5% (n = 49) were Cluster 2 subpopulation. Mixed genotyped infections were also detected but at low frequency . There were 23 P. knowlesi infections that could not be genotyped using the allele-specific PCR assay, and these were excluded in the subsequent analyses.Of 317 P. knowlesi infections from previous studies13, we obtained a total of 1,180 infections throughout the 6-year period from 2014 to 2019, with 68.1% (n = 804) belonging to Cluster 1 subpopulation, 22.8% (n = 269) to Cluster 2 subpopulation and 4% (n = 47) were mixed genotype infections. For subsequent analyses, we excluded 116 infections from the Belaga district since most malaria patients from this district travel to Bintulu hospital instead of Kapit Hospital for treatment. Therefore, a total of 1064 infections from the Kapit and Song districts were analysed . Most infections occurred among the active working-age group between 18 and 64 years old  with a median age of 41 years old, followed by the elderly above 65 years old , school children between 6 and 17 years old , and only four cases among children below 5 years old.From 2014 to 2019, male patients have consistently remained predominant over the years , and the frequencies were consistent throughout the 6-year period, compared to activities conducted > 2 km from the residential areas  of the longhouse in the evening. Patients involved in forest logging activities accounted for 14.6% (n = 155) of the total cases, contributing to the highest number of cases conducted more than 2 km away from the residential areas  and Cluster 2 (n = 245) infections. Patients with Cluster 1 infection showed similar clustering distribution and hotspots pattern to those of the overall P. knowlesi infections  = − 2.14, P = 0.03). This was not observed for Cluster 2 where both groups of activity type contributed equally to knowlesi malaria infections (independent t (8) = − 1.37, P = 0.10).Activities conducted less than 2 km radius within the longhouses and school compounds contributed more cases compared to activities conducted remotely for both clusters (Fisher’s Exact P = 0.03). For Cluster 1 infected individuals, those engaged in activities near the longhouses or within the school compound acquired knowlesi malaria more than those working at the remote forests  compared to females (29%) and occurred remotely in the deep forests for Cluster 2 infections. Cluster 1 infections, however, consisted of an equal ratio of both gender since most of the activities occurred within the radius of longhouses, involving small-scale farming, fishing and poultry rearing.Despite the different proportions of Cluster 1 and Cluster 2 infections, we observed clustering patterns of distribution of 27. Additionally, construction workers were exposed to mosquitoes that feed on wild macaques during forest clearance, especially pig-tailed macaques, increasing the chances in acquiring Cluster 2 P. knowlesi infections. Due to this, the hotspot of knowlesi malaria in this area may be related to the development of the Baleh area because of forest clearance. A similar observation was made in the neighbouring state of Sabah, where predictive analysis showed there was a strong link between deforestation and P. knowlesi occurance27.In recent years, Sarawak has been focusing on reducing the dependence on fossil fuels and non-renewable resources by providing access to sustainable modern energy. Baleh, located approximately 90 km southeast of Kapit town, was selected for the development of a hydroelectric dam in 2015. This resulted in increasing development of infrastructure such as roads, which resulted in forest clearance and altered the habitat of macaques and mosquitoes in the areas36. An early molecular entomological study conducted in Kapit district incriminated Anopheles latens as the vector, and these mosquitoes are predominantly found at forest fringes in farming areas37. Recent entomological surveillance conducted at low zoonotic malaria transmission areas in Betong and Lawas districts showed additional potential vectors such as An. balabacensis, An. donaldi, An. roperi and An. collessi39. Since both macaque species are widespread and have different behavior, it is unknown whether different mosquitoes feed on these macaques at different habitat types or on selected P. knowlesi genotypes. Compared to long-tailed macaques, pig-tailed macaques spend more time in the ground foraging at primary forests and at the fringes of oil palm plantations33. Therefore, comprehensive entomology studies to determine the bionomics and the diversity of Anopheles mosquito species, genotyping of divergent P. knowlesi parasites, combined with studies to map the distribution of the two macaque species are essential to understand the transmission of both genetically divergent P. knowlesi subpopulations.Environmental alteration by humans potentially promotes the presence and abundance of disease vectors13. Human-mosquito-human transmission has been demonstrated under experimental conditions in the 1960s40 and recent studies have demonstrated the presence of viable gametocytes in knowlesi malaria patients so it is possible and may well be occurring42. However, proving that such transmission is currently occurring would be extremely difficult since all the cases are occurring in areas close to habitats of the reservoir macaque hosts.The available evidence strongly suggests that knowlesi malaria is a zoonosis and that the primary hosts are long-tailed and pig-tailed macaquesP. knowlesi infections in the Kapit division are strongly associated with agricultural and forest-related activities. There are significant differences in risk activities between Cluster 1 and Cluster 2 infections, where most cases for Cluster 2 infections are related to deep forest activities. Malaria hotspot analysis provides a useful tool in efforts to control malaria by utilizing time-consistent updates of localities of infection.In conclusion, the current study shows that hotspots of Supplementary Information.


Hymenolepis nana) and Schistosoma haematobium infections could have on the immunoglobulin (Ig) candidate protein of the malaria vaccine GMZ2 levels was evaluated.Malaria and helminthic parasites are endemic in tropical countries, and co-infections might influence host-parasite interactions. In this community-based cross-sectional study, the effect that the presence of soil-transmitted helminths (STH) , STH, and Schistosoma haematobium, respectively. Identification and quantification of the parasite load of STH and S. haematobium were achieved by light microscopy. A polymerase chain reaction was carried out to detect submicroscopic infections of P. falciparum. Plasma levels of GMZ2 specific IgG and its subclasses were quantified by ELISA.Blood, stool, and urine samples were collected from 5-15-year-old children to diagnose H. nana was significantly lower in the mono-infected group with Pf (p = 0.0121) or study participants without infection (p=0.0217). Similarly, the median level of IgG1 was statistically lower in Pf/H. nana group compared to Pf-group (p=0.0137). Equally, the Pf/H. nana infected individuals posted a lower level of IgG1 compared to Pf-group (p=0.0137) and IgG4 compared to the Pf-group (p=0.0144). Spearman rank correlation analyses indicated positive relationships between the densities of H. nana  and S. haematobium .The median level of total IgG in individuals with co-infection with Pf/H. nana infections are associated with reduced GMZ2 specific IgG levels. This study shows the possible manipulation of immune responses by helminths for their survival and transmission, which may have serious implications for vaccine development and deployment in helminth-endemic regions.Hookworm and  The results suggest that hookworm and dwarf tapeworm, a less fancied cestode, infections significantly reduce levels of GMZ2 antibodies in individuals co-infected with malaria parasites and any of these worms. This may compromise the effectiveness of antibody production and antibody-mediated immune response to malaria in individuals with these parasites or the development of GMZ2-mediated immunity against malaria in individuals vaccinated with GMZ2 while infected with any of these parasites. Therefore, the study shows a manipulation of immune responses by these worms for their survival and transmission, which can have profound consequences for the development and deployment of the malaria vaccine in endemic regions.Geographically, malaria and infections caused by worms, including intestinal worms and liver flukes, overlap greatly in distribution, and hence there are instances where these parasites infect the same person simultaneously. Pockets of studies indicate that these worms may have an antagonistic effect on the immune response to malaria when coinfected, while others suggest a protective effect, especially against the severe form of malaria. Therefore, this study looked at how intestinal worms such as dwarf tapeworm, hookworm, Schistosoma spp and soil-transmitted helminths (STH), such as Ascaris lumbricoides, hookworm, and Trichuris trichiura, are highly prevalent in developing countries. These parasites are responsible for more than 40% of worldwide morbidity from all tropical parasitic infections other than Plasmodium spp  responsible for the most severe form of the disease. However, in the human host, Pf and helminths occupy different niches, and any interaction between them may be indirect. This interaction may occur through the influence each of them has on immune responses. Malaria elicits a non-sterile immunity in age and exposure-dependent manner, first against severe malaria and later against uncomplicated malaria after repeated infections  was calculated as the number of parasites per gram of stool, the number of parasite eggs per 10 ml of urine in the case of S. haematobium, and the density of P. falciparum was evaluated as the parasite per microlitre of blood. However, the details of the study site, sample collection, and diagnosis are described elsewhere . The OD values for the test samples were converted into antibody concentration with the standard reference curves generated for each plate using the predetermined concentrations of the serially diluted standards and their corresponding OD values. A four-parameter curve fitting software  was employed in the generation of the standard curve and transformation of the ODs of the samples into concentration units.The study utilized samples collected in Pategi and Lafiagi communities in Kwara State, North Central Nigeria from October 2012 to May 2013, which traversed dry and rainy seasons. The Kwara State Ministry of Health Ethical Committee approved the study, with the ethical certificate reference number being MOH/KS/777/41. Whole blood, stool and urine samples were collected from participants (children between 5 and 15 years) whose parents/guardians gave verbal informed consent after the study procedures, samples to be taken, study benefits, potential risks, and discomforts were explained. In addition, assent was obtained directly from the participating school children. The study participants included only children between the ages of 5 and 15 years who were asymptomatic for malaria and helminthiasis and showed no signs of illness. The parasite burden for STH that included lsewhere . Total Ilsewhere , 10. BriStatistical analyses were performed using the GraphPad Statistical software package version 8.0 . Differences between three or more groups were evaluated by Kruskal-Wallis one way analysis of variance followed by Dunn correction for multiple comparisons as the data was not normally distributed. Spearman rank correlation analysis was used that ranks data points and determines the strength and direction of the relationship between two ranked variables to assess the relationship between parasite load/burden and immunoglobulin levels as the data were not normally distributed. For all tests, the difference between the groups was considered statistically significant at p<0.05.Pf, those with H. nana mono-infection (Hn), those with Hookworm  single infection (Hw), single Schistosoma haematobium infection (Sh), those with coinfections of Pf and Hn., Pf and Sh, Pf and Hw coinfections. However, groups that included multiple infections with only helminths were excluded from the analyses. Of the 450 children, 44 (9.78%), 47 (10.44%), 24 (5.33%), and 157 (34.89%) tested positive for Pf, Hw, Hn, and Sh, respectively. Study participants with Pf/Hw, Pf/Hn, and Pf/Sh are, respectively, 11 (2.44%), 14 (3.11%), and 52 (11.56%), while those with three or more infections are 33 (7.33%). Only 68 (15.11%) children tested negative for all parasites.A total of 450 children were enrolled in this study. The children were 5-15 years with a mean age of 9.5 years. Of these, 220 (48.89%) were females, and 230 (51.11%) were males. For statistical analyses, study participants were grouped as those who were not infected with any parasites at the time of sampling (No infection), those who were infected with only P. falciparum densities during mono-infection and the presence of various helminths were statistically similar  or study participants who were parasite-free at the time of sampling   is significant  statistically differed from the Pf alone  groups . Median levels of the different subclasses of IgG in the various groups of study participants exposed to Hookworm were statistically similar (p>0.05) when compared according to the individual IgG subclass groupings  in dampening the immune response  and PANDORA-ID-Net, funded by EDCTP and the European Union.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.