Plasmodium spp. circulating in the study area were also included in the objectives.Malaria is the fifth leading cause of death worldwide. Pakistan is considered as a moderate malaria-endemic country but still, 177 million individuals are at risk of malaria. Roughly 60% of Pakistan’s population, live in malaria-endemic regions. The present study is based upon the survey of various health care centers in 10 major cities of Northern and Southern Punjab to find out the malarial infection patterns in 2015. The diagnosis, seasonal variations, age and gender-wise distribution of Plasmodium species were amplified, sequenced, blast and the phylogenetic tree was constructed based on sequences using online integrated tool MEGA7.The malaria-suspected patients ‘16075’ were enrolled for malaria diagnosis using microscopy, out of which 925 were malaria positive which were processed for molecular analysis using nested PCR. The 18S rRNA genes of P. vivax (66.7%), P. falciparum (23.7%) and mixed infection by these two species (9.6%) were diagnosed. The same trend  in species identification %age was confirmed from molecular analysis. However, the occurrence of malaria was higher in Southern Punjab (5.5%) as compared to the Northern Punjab (4.0%). The overall malaria percentage occurrence of treatment-seeking patients in all recruited cities of Punjab was 4.9%. The age-group of 1–20 and males among genders were more affected by malaria. The comparison of different seasons showed that the malaria infection was at a peak in Summer and post-monsoon.The 364 cases recruited from Northern Punjab with the highest incidence in Rawalpindi (25.5%) and lowest in Chakwal (15.9%). From Southern Punjab 561 cases were enlisted Rajanpur (21.4%) maximum and lowest from Multan and Rahim Yar Khan (18%). The slide positivity rate, annual parasite incidence, and annual blood examination rates were 5.7 per 1000 population, 0.1, and 0.2% respectively. The only The incidence of malaria was high in the flood infected rural areas of Southern Punjab, Summer, and post-monsoon. The age group (1–20) and gender-wise males were more affected by malaria. Plasmodium species in human; P. vivax, P. falciparum, P. ovale, P. malaria and P. knowlesi (zoonotic infection). Regardless of massive and costly malarial control measurements have been taken over many decades but still, it has re-emerged as a serious health problem in Asia. Sixty percent population of Pakistan lives in malaria-endemic regions. Plasmodium vivax contributes 81.3%, P. falciparum 14.7%, and mixed-species 4% in the malaria burden of Pakistan [Malaria is the globally largest vector born disease with over 200 million clinical cases happening every year. Malaria has been associated with 0.6 million deaths among children and pregnant women annually. The world is currently not on track to achieve the milestones of 2020 of WHO Global Technical Strategy (GTS) of malaria 2016–2030. The milestone of 2020 is to reduce malaria death and disease by 40%. There is a continuous increase in malaria cases from 2015 to 2018. The number of malaria cases globally increases as 214, 217 and 219 million from 2015, 2016 and 2017 respectively [The endemicity of malaria varies in Punjab being the land of five rivers Sutlej, Chenab, Beas, Ravi, and Jhelum generate a major breeding ground for malaria vector. Punjab is the most populous province of Pakistan with an estimated population of 110,012,442 according to the 2017 census of Pakistan. It experiences all four types of seasons; spring (March to May), summer (June to September), autumn (October to November) and winter (December to February). The onset of the southwest monsoon in Punjab most likely takes place from the month of June till September but the weather pattern in Pakistan has been irregular since the last few decades. Spring monsoon has either missed or has caused heavy rain over the area that results in floods. The month of June and July are extremely hot. This domineering heat is interrupted by the rainy season in August. The hardest part of the summer season is then over, but cooler weather does not come until late October .Punjab’s plains are low-lying, wet and often swamped. Malaria is seasonal and unpredictable in this province and its epidemics repeated at about 8-year intervals. Variation in malaria transmission from year to year is due to the floods in the Southern Punjab and heavy rainfall during monsoon in Northern Punjab. However, the repeated changes in climate patterns are linked with variation in the malaria transmission pattern .P. falciparum is high among the Afghan refugees than the local population. Several epidemiological studies have revealed that there was a 24 to 36% increase in malaria cases in Pakistan due to the influx of Afghan refugees [P. vivax infection is restrained to two peaks per year, the main peak was in late spring as a result relapses of earlier infections and additional peaks occurred in Summer and Autumn by recent transmissions [P. vivax, P. falciparum malaria showed an increasing correlation with August–September temperatures [Malaria is more prevalent in rural areas due to low socioeconomic conditions . The prerefugees , 8. The refugees . The P. missions . In conteratures . The wideratures .P. falciparum infection in the last decade. The increase of P. falciparum infection across the country can be associated with chloroquine drug resistance [The National Malaria Control Program has reported a six-fold increase in sistance  also in sistance . The malsistance . Besidessistance , 15.Plasmodium species is necessary for its treatment and drug formulation. The primary method is microscopy however the most advanced, proficient, sensitive and precise method for the detection of Plasmodium species is molecular analysis using Polymerase chain reaction (PCR). The PCR analysis can identify Plasmodium species and mixed infections even at low parasitemia [Plasmodium species are also identified circulating in the studied area and their individual incidence is calculated statistically. It will be a great contribution in the epidemiology of malaria in Pakistan and helpful in further malaria control strategies especially in educating people and drug formulation.The correct diagnosis of asitemia , 17. It asitemia . The preThis study was part of Quaid-i-Azam University Research Fund Pakistan project aiming to determine the in-vivo effect of medicinal plant extracts and nanoparticles on malaria parasite. A cross-sectional epidemiological study of malaria was conducted from January to December 2015, recruited 16,075 suspected malaria cases in different hospitals  detection.A total of 925 microscopically positive blood samples were selected for PCR confirmation and delivered to Quaid-i-Azam University Parasitology lab in the cold chain. The DNA was extracted by using the Gene Jet Genomic DNA Purification Kit , according to the manufacturer’s instructions and then stored at − 20 °C. Human malarial parasitic DNA was detected through nested polymerase chain reaction (PCR) high sensitive detection method was adopted from Snounou et al.  using sm2 4 μL, dNTPs 1 μL, 2 μL of each forward and reverse primer, Taq polymerase 0.5 μL and 3 μL DNA template. For the 2nd round, 4 μL of the 1st round PCR product was used as a template with the same quantity of master mix as mentioned in the 1st round. A plasmid containing DNA from Plasmodium species were obtained from MR4  for the amplification of all five Plasmodium species. Few malaria positive samples were also collected from different sentinel sites of the Directorate of Malaria Control Program for positive control whereas, healthy blood samples were used as a negative control. The visualization of amplified PCR products was conducted by electrophoresis using 1.5–2.0% agarose gel stained with ethidium bromide and visualized under UV-trans-illuminator . The smear-free amplified PCR products were purified using a gene jet PCR purification kit  (cat# K0701) and sent to Korea (macrogen Inc.) for sequencing. The evolutionary tree was constructed using Mega 7 software.The amplification of the 18S rRNA gene was carried out using the primers and nested cycling conditions reported by Snounou et al. . In a 1sThe count of malarial incidences was analyzed in relation to the age and gender of patients and the season of malaria occurrence. Initial data exploration revealed that count data is zero-inflated and over-dispersed (mean = 9.64 was much lower than variance = 310.31). We, therefore, tested the following three models in R software :M1: Zero-inflated negative binomial regression with random effect of season.Zeroinfl .M2: Zero-inflated negative binomial regression without random effect.Zeroinfl ).M3: Simple negative binomial regression.glm.nb .p = 1.1672e-06) over a zero-inflated negative binomial model. Therefore, the results of the negative binomial model are used in this study. Data visualization was performed using the ‘ggplot2’ package in the R program. The script for comparison model is given in an Additional file For comparison of the models, the Vuong test was used, which suggested that the negative binomial model was a significant improvement , fair (0.2 to 0.4), moderate (0.40 to 0.6), good (0.61 to 0.80), and very good (0.8) in order to check the strength of agreement between PCR and microscopic examination. The Cohen ‘Kappa tests were conducted on the SPSS V 19.0.Out of 16,075 entire suspected cases, 925 were positive and 15,150 were negative. The overall slide positivity rate (SPR) was 5.7% in all recruited cities of Punjab however; the SPR was higher in Southern Punjab (6.1%) as compared to the Northern Punjab (5.3%). The SPR within the recruited cities of Northern Punjab was highest in Jhelum lowest in Chakwal i.e. 6.8 and 3.1% respectively. In contrast among observed cities of Southern Punjab, the SPR was maximum in Rajanpur (7.1%) minimum in Dera Ghazi Khan (5.4%). The API was 0.1 per 1000 population in all recruited cities., 1.2 and 0.06 per 1000 population respectively. The ABER was 0.2 in Punjab and it was greater in Southern Punjab as compared to the Northern Punjab 0.26 per 1000 population > 0.2 per 1000 population. The ABER within Northern Punjab was high in Chakwal and minimum in Gujranwala i.e., 1.3 and 0.1% respectively. The ABER in Southern Punjab was highest in Rajanpur (1.7%) lowest in Multan (0.2%)  as compared to the Northern Punjab (0.09 per 1000 population). The API within Northern Punjab was high in Jhelum and it was equal to API in Chakwal 0.4 per 1000 lowest in Gujranwala 0.04 per 1000 population. Among the analyzed cities of Southern Punjab, the API was maximum in Rajanpur and minimum in Multan i.e.. Autumn  had a higher rate of incidences as compared to Spring and Winter with expected incidence  of − 0.5671 and − 2.1374, respectively.The negative binomial regression analysis Table  indicateDistribution of malaria case with respect to age and gender are given in the additional file. P. vivax had an expected log (incidence) of 1.0308 higher than that of P. falciparum, however, mix infections are − 0.8562 lower than P. falciparum  of 0.8466 higher than that of females holding other variables constant with 71.78% malaria cases. The n = 617) were P. vivax 23.7% (n = 219) were P. falciparum and 9.6% (n = 89) were mixed infections out of 925 recruited cases (Table n = 494) as P. vivax, 18.7% (n = 173) P. falciparum and 12.7% (n = 119) as mixed species, whereas 15.0% (n = 139) were PCR negative cases. However, no case of P. ovale and P. malariae was found both through microscopy and PCR  were microscopically positive for P. vivax, 19% (n = 69) were P. falciparum and 6.5% (n = 25) had mixed species out of 364 cases. The PCR results showed that 50.3% (n = 183) were of P. vivax, 14.8% (n = 54) P. falciparum, 11% (n = 40) mixed species and 24% (n = 87) did not amplify for any species  P. falciparum and 11.4% (n = 64) of mixed species. The PCR results showed that 55.4% (n = 311) were of P. vivax, 21.2% (n = 119) P. falciparum, (14.1%) (n = 79), mixed species and 9.3% (n = 52) PCR negative (Table In Northern Punjab74.2% (Plasmodium species was good (Kappa = 0.79). However, for the detection of P. vivax the agreement between microscopy and PCR was fair (Kappa = 0.38). But for P. falciparum and mixed infection, it was moderate  respectively.The coincidental adjustment of kappa statistics specified that overall agreement in the presence or absence of , 5.0% followed by Gujrat 4.9%. The malaria prevalence was lowest in Chakwal i.e. 2.3%. Among the studied cities of southern Punjab, the prevalence followed the pattern Rajanpur (6.7%) > Bahawalpur (5.3%) > Rahim Yar Khan (3.1%), Multan (2.1%) > Dera Ghazi Khan (2.9%)  as compared to the Northern Punjab (4.0%). Within Northern Punjab, malaria occurrence was higher in Rawalpindi i.e.P. vivax and P. falciparum taken from Northern and Southern Punjab two from each site P. vivax and one of P. falciparum  was shown next to the branches. The tree was drawn to scale, with branch lengths in the same units as those of the evolutionary distances used to infer the phylogenetic tree. The evolutionary distances were computed using the p-distance method and it was in the units of the number of base differences per site. The analysis involved 21 nucleotide sequences. All positions containing gaps and missing data were eliminated. There was a total of 85 positions in the final dataset. Evolutionary analyses were conducted in MEGA7. One DNA sequence of P. vivax and P. falciparum showed the closest relationship with the P. falciparum partial sequence of 18S ribosomal RNA gene from Brazilian Western Amazon. Another sample of P. vivax from Northern Punjab showed the closest association with the P. vivax with a partial sequence of 18S rRNA genes from Korea. However, one sample both of P. vivax and P. falciparum from Northern and southern Punjab and one sample of P. vivax from northern Punjab showed association with P. vivax of Yunnan Province  in two zones of Punjab was 5.7%. However, the SPR was high in Southern Punjab (6.1% > 5.3%) as compared to Northern Punjab. Mahmood et al.  studied P. vivax in post-monsoon season (September through August) which is slightly prolonged from the previous study conducted by Prybylski et al., [P. vivax was predominant in earlier of the transmission season in July through August. This seasonal shift in P. vivax predominance and suppression of P. falciparum by P. vivax is believed to be related to the greater variation in the climate of Punjab from last decade due to global warming and high production of gametocytemia by P. vivax in post-monsoon. Overall malaria began to increase in the spring season i.e., from March and April. These seasonal variations prolonged the summer and increased the highly favorable environmental conditions for mosquitoes breeding that consequently supported the malaria influx in late summer too. However, towards December to February (coldest season) lowest malaria cases occur due to a change in climate, such change in temperature and rainfall patterns unstable the vector dynamics. The age-wise distribution of malaria was also evaluated in the present study. The age of all recruited cases in both genders was ranged between 1 to 78 years. The high incidence was observed among the age group of 1–20 > 21–41 than among 41–60-year age groups. However, the distribution of malaria parasites was low in high age groups. It indicates that the 1–20 age group is highly infected due to low acquired immunity and exposure to vector because of higher non-resting outdoor activities in this age group. Another major factor for highest infection in age group (1–29) is that the major proportion (64%) of total population of Pakistan lies in this age group. The gender-wise distribution indicated that Plasmodium infection was dominant in males than females in both zones of Punjab. In the present study, 71% males and 39% females were infected. The females in the Southern Punjab (30.5%) were comparatively high as compared to the Northern Punjab (27.5%). The possible reason may be the role of males in agricultural activities. Khattak et al. [In order to prepare for and implement integrated malaria control intervention, it is imperative to understand the seasonality of malaria parasitemia. The outcome of this study confirms that malaria parasitemia in Punjab fluctuates with seasonality variation in climate. In this study, we observed new seasonal patterns by observing the peak prevalence and density of  et al.,  under a k et al. , 29, whiP. vivax, 23.7% P. falciparum and 9.6% were mixed infections out of 925 recruited cases. However, molecular analysis showed that 53.40% as P. vivax, 18.7%, P. falciparum and 12.7% as mixed species and 15.3% were PCR negative cases. According to the microscopy the 90 samples were diagnosed as mixed infection, but PCR results showed 119 samples found to have a mixed infection. Many species that were diagnosed as P. vivax or P. falciparum microscopically had diagnosed as a mixed infection through PCR. The present findings also revealed that mixed infection was high in Southern Punjab as compared to Northern Punjab. Sheikh et al. in 2005 [P. vivax and 30.7% P. falciparum infection. Likewise, Khattak et al. [P. vivax samples were 76%, P. falciparum were 18% and mixed infections were 6%. However, they reported 73% P. vivax, 22% P. falciparum and 5% mixed infection in Punjab province and covered only five cities. While in present study, out of 10 major cities of Punjab highest incidence were found in Rawalpindi 25.5% (n = 93) of Northern zone and Rajanpur 21.4% (n = 120) from Southern Zone. Ahmed et al. in 2013 [P. vivax as 99.4% and only 0.53% due to P. falciparum. Whereas, present study revealed 66.7% as P. vivax, 23.7% P. falciparum and 9.6% mixed infection  by microscopy. The molecular analysis confirmed the presence of the same species with independent and mixed infection although there was a difference in occurrence e.g. P. vivax: 53.4%, P. falciparum: 16.70% and mixed infection: 12.9%. However, the 139 (15.2%) microscopically positive samples did not amplify with the PCR analysis that may be due to the false-positive results of microscopy or may be due to the DNA damaged by anticoagulant used in EDTA tubes. In contrast, high percentage of P. falciparum to P. vivax (65% vs 35%) among children positive for malaria has been recorded at Baqai medical university hospital Karachi, Pakistan in 2002 [P. falciparum (88.5%) as compared to P. vivax (9%) out of 348 recruited malarial patients in Karachi, Pakistan. Likewise, in Multan, P. vivax and P. falciparum were observed in a percentage ratio of 60.5:37.2 respectively [Plasmodium species from Southern to Northern areas of Punjab province Pakistan, since P. vivax infection is becoming higher than P. falciparum. It may be due to decrease in temperature and humidity conditions from Southern to Northern Punjab.The species-wise distribution of malaria, according to the microscopy showed 66.7%  in 2005  investig in 2013  also con in 2002 . Mahmood in 2002  reportedectively . The preP. vivax had a high occurrence except Bahawalpur showed P. falciparum as predominant species. Similarly, in Northern Punjab P. vivax had the highest prevalence and Chakwal was accounted for the highest mixed infection. The recent flee of IDPs  from high malaria prevalence areas such as FATA [P. vivax in Northern Punjab. The phylogenetic kinship of P. vivax and P. falciparum isolates is rather not restricted to one region but seems to be widespread across different geographical regions of the world. The isolates of P. vivax from Northern and Southern Punjab showed homology to the P. vivax isolates of Yamen and India. All isolates of P. falciparum were clustered together and showed homology of P. falciparum isolates from Yunnan, Africa, and India. Similar origin of P. falciparum was observed by Joy et al. [P. vivax showed a more diverse population than P. falciparum. A significant rise in temperature and humidity result in an increase of vector breeding rate and the vectoral capacity that may enhance the Plasmodium infection and its re-emergence [Plasmodium sp. However, its accuracy of the results mostly depends upon the experience of technicians along with other factors such as slide staining pattern, parasite level in the blood and microscope condition also influences the results. PCR is used for the most accurate diagnosis of all the five species of Plasmodium, but it is expensive than microscopy. The epidemiological study of malaria conducted in the Orakzai agency showed a higher positive result of microscopy as compared to PCR [With respect to socioeconomic norms, WHO (2010)  reported as FATA –37 and S as FATA  to Northy et al. . Howevermergence . The micd to PCR , 37. Shad to PCR  conducted to PCR , PCR basPlasmodium sp. occurrence in this study exposed that malarial endemicity was highest in summer and post-monsoon season. It might be because of rice paddies and sugarcane cultivation are at peak in this season, which increases the rate of mosquitoes breeding. Along with the effect of seasonal variations on malaria incidence, other contributing factors which may increase malaria endemicity are like misdiagnosis by untrained microscope technician, presumptive treatments, extensive agriculture activities, improper waste management, inadequate health care facilities in remote areas, low socioeconomic conditions and lack of malaria control intervention strategies. The overall incidence rate of malaria in Southern Punjab found to be high as compared to Northern Punjab may be due to the flood in Southern Punjab. P. vivax was found to be more dominant as compared to P. falciparum. The mixed infection is also very high in Punjab. Pakistan is facing many problems and challenges in the prevention and control of malaria, including misdiagnosis, scarcity of diagnostic facilities, and the use of presumptive treatment. Facts and figures about the burden and species distribution of malaria are acute for guiding national and provincial efforts for effective control measures.The malaria control remains a great challenge in developing countries like Pakistan due to agricultural activities; prolong the monsoon season and frequent floods. The relationship between seasonality and Plasmodium species infection and the monitoring of patients receiving anti-malarial treatment. The growing malarial incidence annexed new ecotypes twisted by, industrial growth, and urban development resulting and the green revolution in model shifts towards man-made malaria. The special attention should be given by the government in the flood infected areas of Southern Punjab and many areas of Sindh in order to eradicate malaria from these regions.Therefore, it is essential to improve the accuracy of malaria diagnostic techniques specially to determine species-specific and low parasitemia and to assist with diagnostic method procurement decisions in a more professional way. This would also ease the calculation of the ratio of mixed Additional file 1. Location (coordinates) and names of malaria sample collection sites in the province of Punjab.Additional file 2. Performs used for malaria sample and secondary information collection from the suspected cases of malaria.Additional file 3. A parent or gaurdian approval form used to collect malaria blood samples from underage <18 year old malaria patients.Additional file 4. The script used for Zero-inflated Negative Binomial Regression model in R-3.4.4 statistical software.Additional file 5. Number and percentage of malaria cases in whole Punjab with respect to gender and age.


Plasmodium continues to be a major health problem worldwide. The unicellular Plasmodium‐parasites have the unique capacity to infect and replicate within host erythrocytes. By expressing variant surface antigens Plasmodium falciparum has evolved to avoid protective immune responses; as a result in endemic areas anti‐malaria immunity develops gradually over many years of multiple and repeated infections. We are studying the role of Plasmodium falciparum erythrocyte membrane protein 1 (PfEMP1) expressed by asexual stages of P. falciparum responsible for the pathogenicity of severe malaria. The immunopathology of falciparum malaria has been linked to cyto‐adhesion of infected erythrocytes to specific host receptors. A greater appreciation of the PfEMP1 molecules important for the development of protective immunity and immunopathology is a prerequisite for the rational discovery and development of a safe and protective anti‐disease malaria vaccine. Here we review the role of ICAM‐1 and EPCR receptor adhering falciparum‐parasites in the development of severe malaria; we discuss our current research to understand the factors involved in the pathogenesis of cerebral malaria and the feasibility of developing a vaccine targeted specifically to prevent this disease.Malaria, a mosquito‐borne infectious disease caused by parasites of the genus  The ability of P. falciparum‐infected erythrocytes (IEs) to adhere efficiently to host vascular receptors sets this parasite aside from the other malaria parasites infecting humans, and is generally considered an important reason why P. falciparum malaria is particularly dangerous.Infection with Plasmodium falciparum erythrocyte membrane protein 1 (PfEMP1), encoded by the clonally variant var gene family with approximately 60 members per parasite genome. This review focuses on PfEMP1, the putative role of this antigen family in the development of one of the most severe forms of malaria called cerebral malaria (CM) and in acquired immunity to CM, and finally on the prospect of a PfEMP1‐based vaccine to prevent this often fatal complication. Before discussing each of these aspects it is necessary to recapitulate briefly the parasite life cycle, as it is important for appreciating the sections that follow.IE adhesion is called sequestration when the IEs stick to tissue‐bound receptors, rosetting when they stick to uninfected erythrocytes, and clumping when the IEs stick to each other. It can lead to circulatory disturbances, vascular occlusion, and inflammation. In all cases, the IEs interact with host receptors via members of parasite‐encoded antigens displayed on the IE surface. These antigens belong mainly—if not exclusively—to products of several multigene families. Prominent among them—and by far the best studied—is 1.1Plasmodium falciparum has a complex life cycle that involves two hosts (humans and Anopheles spp. mosquitoes), and several developmental stages in each host  schizont that consists of at least 30 000 daughter parasites. These, now called (pre‐ or extraerythrocytic) merozoites leave the infected hepatocyte and enter the blood circulation. The merozoites rapidly infect erythrocytes, an event that marks the beginning of the intraerythrocytic multiplication cycle. This part of the life cycle continues until the infection is controlled by either immunity or chemotherapy, or until the host dies. Each round of the intraerythrocytic cycle lasts approximately 48 hours. During each, the newly invaded merozoite rapidly transforms to a trophozoite  that undergoes three to five mitotic divisions, resulting in a schizont. At the end of the intraerythrocytic cycle, the IE ruptures and the released (erythrocytic) merozoites rapidly invade new erythrocytes. Some of the newly invaded merozoites develop into male or female gametocytes rather than continuing the asexual multiplication cycle. The gametocytes do not divide, but remain inside the erythrocyte until taken up by a blood‐feeding mosquito, where sexual reproduction and further asexual multiplication steps complete the parasite life cycle.t Figure . The hum2The expression of PfEMP1 is largely , encoded by approximately 60 two‐exon 0 kD, encP. falciparum EBA‐175 adhesive domains and to the Duffy‐binding proteins of P. vivax and P. knowlesi.The PfEMP1 CIDR domains are characterized by conserved cysteine‐rich motifs,ups), and direction of transcription of the var genes encoding them.P. falciparum 3D7), Group B (22 genes), and Group B/A var genes (4 genes) are all found in the subtelomeric regions of chromosomes. Group A genes are transcribed toward the telomere, whereas Group B and B/A var genes are transcribed toward the centromere  and Group B/C var genes (9 genes) are typically found in internal regions of chromosome 4, 7, 8 and 12.Despite the sequence variability, PfEMP1 proteins can be grouped according to their chromosomal location, upstream promotor sequence .Most PfEMP1 appear to be elongated and are rigid molecules with a zigzag shape and a length of about 30 nm,2.2The primary function of the PfEMP1 proteins is to mediate adhesion of IEs to host receptors in the vasculature.Many different PfEMP1 proteins appear to have specificity for the same receptor, and this to some extent corresponds to the structurally defined PfEMP1 groups and domain subclasses mentioned above.2.3P. falciparum parasites and is a major determinant of the characteristic chronicity of untreated infections.P. falciparum parasites are complex, and will not be described here, as they have been recently reviewed in detail elsewhere.var genes.Aside from an exception of unresolved biological significance,2.4The expression of PfEMP1 on the IE surface is confined to membrane protrusions called knobs.Plasmodium helical interspersed subtelomeric] protein family .53, 56,The knobs appear on the IE surface approximately 16 hours postinvasion, peak in density about 20 hours later, followed by a slight decrease in density toward the end of the intraerythrocytic 48‐h cycle.P. falciparum parasites to adequately remodel the erythrocyte cytoskeleton and display PfEMP1 in these aberrant erythrocytes.Altogether, these findings suggest that the overall IE adhesiveness is the net result of which PfEMP1 is expressed , how much of it is expressed (quantity), and how it is expressed (topology). This conclusion is supported by studies linking the protective effects of haemoglobinopathies such as HbC, HbS, and α‐thalassemia to an impaired ability of 3P. falciparum malaria (CM) is defined by the World Health Organization (WHO) as deep and unarousable coma that persists for more than 1 hour after a seizure, irrespective of anticonvulsant medication, in a patient with peripheral P. falciparum parasitemia and without another cause of encephalopathy.P. falciparum develop CM, which has a very high mortality despite treatment, with up to 75% of deaths occurring within the first 24 hours postadmission.Cerebral P. falciparum infection. Several studies have shown seasonal and transmission intensity‐dependent differences in the frequency of CM,In sub‐Saharan Africa, CM mostly affects children under 5 years of age who have only partial acquired immunity to There are significant differences in the pattern of vital organ dysfunction between African children and Southeast Asian adults with CM.In the following sections, we will focus on CM as it occurs among children in Africa because this is the population most affected, and also because most of the available knowledge about CM stems from studies of African children Box .Box 1P. berghei ANKA (PbA) is a popular in vivo model to dissect the mechanisms of CM. PbA infection can induce a condition known as experimental cerebral malaria (ECM), which recapitulates some—but certainly not all—features of CM. This has led to considerable scepticism regarding the utility of this murine model.A detailed analysis of the intracerebral pathogenesis and pathology in CM comes primarily from postmortem studies. For this reason, infections of various strains of mice with + cells appear central to ECM pathogenesis. + T‐cell accumulation in human CM.In both humans and mice, the clinical signs of CM progress from seizures, ataxia, and paralysis to coma and eventually death.ECM is characterized by a prominent pro‐inflammatory cytokine response with high levels of IFN‐γ and TNF‐α, which results in upregulation of activation markers including ICAM‐1, VCAM‐1, and E‐selectin.3.1Retinopathy, characterized by retinal hemorrhages, papilledema, retinal whitening, and vessel color changes, is the most specific clinical diagnostic sign of CM,Pediatric CM can also be divided into two types based on clinical and pathological findings. CM1 (25% of cases) is characterized by IE sequestration in the cerebral microvasculature, rapid disease progression, limited organ pathology, and high splenic parasitemia. CM2 is characterized by more prolonged illness and parasite proliferation, with evidence of vascular brain pathology alongside endothelial dysfunction, activation of coagulation, ring hemorrhages, fibrin‐platelet thrombi, and accumulation of monocytes in addition to sequestered IEs.Increased intracranial pressure with brain swelling and perivascular edema are strongly associated with CM severity.3.2The BBB is vital for normal brain function and constitutes a physiological barrier that separates the brain and the cerebrospinal fluid from the rest of the body.The BBB components include microvascular endothelial cells forming a continuous barrier through tight junctions, a basement membrane, pericytes, and astrocytes that are in direct contact with neurons and microglia. This composition is critical to minimize local inflammation and neuronal damage.Disruption of the BBB is common in diseases of the central nervous system.3.3P. falciparum malaria and correlates with disease severity in general and CM in particular.Endothelial inflammation is a characteristic feature of Endothelial cells derived from Malawian children with CM have been shown to express particularly high levels of parasite and platelet receptors, to produce many endothelial microvesicles, release high levels of pro‐inflammatory cytokines (including TNFα and IFNγ), and to be highly prone to undergo apoptosis.Activated brain endothelial cells are known to express high levels of a number of potential IE receptors , exocytose Weibel‐Palade bodies, release microvesicles, vascular endothelial growth factor (VEGF), and soluble cell adhesion molecules , and to show breakdown of tight junctions.von Willebrand Factor is synthesized by the endothelial cells, and some of the synthesized vWF is constitutively secreted into plasma, but most is stored within Weibel‐Palade bodies and only secreted following activation of the endothelial cell.4P. falciparum parasites display PfEMP1 molecules on the surface of the erythrocytes they infect. From about 16 hours postinvasion, these high‐molecular weight variant parasite proteins efficiently mediate adhesion of the IEs to a range of host receptors,P. falciparum infection. This hypothesis has been confirmed in the case of placental malaria, where the selective accumulation of IEs in the intervillous space is mediated by VAR2CSA‐type PfEMP1 with affinity for placental CSA.P. falciparum malaria, not least CM.As mentioned above, 4.1IEs adhere to host endothelial receptors in the postcapillary venules of a number of organs, such as lungs, liver, intestine, brain, and the placenta.var genes encoding PfEMP1 proteins from Group A and B/A.P. falciparum parasites isolated from African children with CM bind preferentially to ICAM‐1 in vitro.Many studies have reported links between severe malaria (including CM) in children and infection with parasites transcribing P. falciparum 3D7 PfEMP1 PFD1235w,pfd1235w gene in parasites from Ghanaian malaria patients, inspired by the link between PFD1235w and severe malaria.3N[E]GG[P/A]xYx27GPPx3H).IE adhesion to ICAM‐1 is mediated by PfEMP1 variants that can also bind to either EPCR or CD36.var genes.P. falciparum parasites obtained from African children and Indian adults with severe malaria—including CM—transcribe DC8‐ and DC13‐encoding var genes at high levels.var genes encoding Group A PfEMP1.Endothelial protein C receptor is the cognate receptor for PfEMP1 proteins containing domain cassette DC8 or DC13.P. falciparum isolates can bind to it.As mentioned above, not all PfEMP1 variants capable of binding ICAM‐1 also bind EPCR. Indeed, all but oneCombining the above evidence, the adhesion phenotype that is most clearly related to CM is IEs expressing Group A PfEMP1 proteins (including DC4) that allow concomitant binding to both ICAM‐1 and EPCR (“double binders”).4.2P fragile‐infected monkeys.P. falciparum,Infected erythrocytes do not only adhere to the endothelium, but also to surrounding uninfected or infected erythrocytes. The former type of such aggregates, called rosettes, were first reported in the Infected erythrocytes isolated from patients with CM have been reported to form rosettes at significantly higher rates than IEs from patients with uncomplicated malaria.2‐macroglobulin as another important cofactor of rosetting,The receptor specificity of the PfEMP1 variant expressed is an obvious determinant of whether PfEMP1‐dependent rosetting occurs or not. However, soluble plasma factors that can bind to PfEMP1 may also be of pathologic significance. Thus, IE rosetting in vitro generally  requires the presence of plasma or serum in the assay, and several components have been implicated.IEs can also bind to other IEs via platelets (thrombocytes); a phenotype referred to as clumping.4.3Erythrocytes normally flow down the central line of a blood vessel,4.4P. falciparum malaria, the pathophysiology of the disease is only partly understood.In spite of the very significant morbidity and high mortality of cerebral The protein C pathway is a crucial anti‐coagulant and anti‐inflammatory regulator of thrombin production during clot formation.It has been proposed that this delicate system of checks‐and‐balances may be upset by IEs adhering to EPCR, thereby preventing physiologically appropriate activation of protein C.Brain microvascular endothelium may be particularly susceptible to the disruption of the protein C pathway by EPCR‐adhering IEs because EPCR is expressed at low levels at this site as opposed to the high expression seen in arteries and veins.The impact of EPCR‐binding IEs on the protein C pathway proposed above is consistent with features of CM pathogenesis, but most studies have found this IE adhesion phenotype to be associated with severe malaria in general, rather than with CM specifically.5P. falciparum parasites, susceptibility to clinical malaria is inversely correlated with age. Antibodies to parasite antigens on the surface of IEs are important, even decisive, determinants of this relationship, including the gradually decreasing risk of developing severe malaria such as CM.In areas with stable transmission of 5.1P. falciparum malaria hinges on the idea that the infecting parasites adapt to pre‐existing and developing immunity by switching to variants that are not recognized by specific antibodies.The variant‐specific, PfEMP1‐centric hypothesis of susceptibility to, and acquired immunological protection against, var genes and acquisition of PfEMP1‐specific IgG follows this pattern, and shapes what PfEMP1 variants are compatible with parasite survival in a given semi‐immune host.var gene transcription in children with limited pre‐existing immunity and severe disease is skewed toward Group A,var genes in Group B and C, encoding PfEMP1 variants associated with uncomplicated disease gradually increases.P. falciparum infection, and the acquisition of immunity to that particular form of severe malaria, as both depend on a particular type of PfEMP1 called VAR2CSA.P. falciparum malaria, including CM.Variant‐specific immunity is indeed acquired in the orderly fashion predicted by the above theory. Antibodies to relatively conserved (“common”) parasite antigens and protection from severe malaria are acquired first, followed by antibodies to more variant antigens associated with uncomplicated disease, and eventually by antibodies recognizing very diverse (“rare”) antigens expressed on the surface of IEs obtained from carriers of asymptomatic/subclinical infections.P. falciparum, and this acquisition is associated with protection from severe malaria, including CM.IgG antibodies to the Group A, DC4‐containing PfEMP1 that can mediate IE adhesion to both ICAM‐1 and EPCR are acquired early in life in areas with stable transmission of The degree of IgG cross‐reactivity that transcends clone‐specific variability in key antigens is of obvious importance. Encouragingly, naturally acquired antibodies capable of inhibiting the interaction between ICAM‐1 and many ICAM‐1‐binding DBLβ domain variants were recently observed.5.2P. falciparum. While this goal is elusive at the present time, vaccines that prevent severe malaria illness, particularly in children and pregnant women, could constitute an alternative or interim strategy.An ideal malaria vaccine would elicit immunity that prevents infection of humans and leads to transmission elimination and global eradication of Current efforts to develop VAR2CSA‐based vaccines to prevent placental malaria is the most advanced example of a PfEMP1‐based approach to malaria vaccination, as several such vaccines are currently in clinical trials.However, many questions remain unanswered. Aside from the uncertainty regarding the feasibility of making a vaccine with the desired qualities, it is unclear whether inhibition/reversal of ICAM‐1/EPCR‐specific IE adhesion would be sufficient to prevent CM. It is similarly unclear to what extent acquired clinical protection from CM requires neutralizing (IE adhesion‐blocking) antibodies, as opposed to opsonizing antibodies leading to preferential phagocytic and/or cytotoxic removal of IEs expressing particularly pathogenic PfEMP1 variants.While efforts to overcome the obstacles in the development of a PfEMP1‐specific vaccine to protect against CM continue, other interventions should also be pursued. Unfortunately, adjunctive therapies , aimed at protecting against CM‐related brain damage and neuronal injury, have so far been unsuccessful.6P. falciparum parasites and the humans they infect is progressing at a rapid pace. This is not least so for the tissue‐, organ‐, and receptor‐specific adhesion of IEs that act as major contributors to the pathogenesis of P. falciparum malaria, including the development of severe complications such as CM.Our understanding of the molecular basis of the interactions between P. falciparum parasites have evolved a range of other strategies to evade PfEMP1‐specific immunity. Examples are antigen topology and cloaking, interference with antigen presentation, and subversion of soluble host molecules.The critical importance of PfEMP1 for parasite survival makes these antigens major immune targets, and acquisition of PfEMP1‐specific antibodies indeed appears to be a central component of naturally acquired protection. Emulating and accelerating these responses through vaccination would therefore seem obvious. However, the flipside is that the importance of PfEMP1 to the parasites applies a strong selective pressure on them to evolve mechanisms to evade protective, PfEMP1‐specific immunity. The most prominent example of this is clonal antigenic variation, which undoubtedly evolved to delay and frustrate the acquisition of PfEMP1‐specific protective antibodies, thereby enabling chronic infections.P. falciparum malaria constitutes to health and to economic progress and equality, we are obliged to continue this research.In conclusion, much has been learnt from years of research on CM pathogenesis and immunity, although a lot remains to be known. Because this malaria complication is such an important part of the intolerable obstacle that The authors declare no competing interests.


Plasmodium vivax-like autochthonous human cases has been reported in remote areas of the Atlantic Forest in the past few decades in Rio de Janeiro (RJ) state, including an outbreak during 2015–2016. The singular clinical and epidemiological aspects in several human cases, and collectively with molecular and genetic data, revealed that they were due to the non-human primate (NHP) parasite Plasmodium simium; however, the understanding of the autochthonous malarial epidemiology in Southeast Brazil can only be acquired by assessing the circulation of NHP Plasmodium in the foci and determining its hosts.Although malaria cases have substantially decreased in Southeast Brazil, a significant increase in the number of Plasmodium infections via molecular and microscopic techniques.A large sampling effort was carried out in the Atlantic forest of RJ and its bordering states  for collecting and examining free-living NHPs. Blood and/or viscera were analyzed for Alouatta clamitans) were the only species found infected. In RJ, 26% of these monkeys tested positive, of which 17% were found to be infected with P. simium. Importantly, specific single nucleotide polymorphisms–the only available genetic markers that differentiate P. simium from P. vivax–were detected in all P. simium infected A. clamitans despite their geographical origin of malarial foci. Interestingly, 71% of P. simium infected NHPs were from the coastal slope of a mountain chain (Serra do Mar), where majority of the human cases were found. Plasmodium brasilianum/malariae was initially detected in 14% and 25% free-living howler monkeys in RJ and in the Espírito Santo (ES) state, respectively. Moreover, the malarial pigment was detected in the spleen fragments of 50% of a subsample comprising dead howler monkeys in both RJ and ES. All NHPs were negative for Plasmodium falciparum.In total, 146 NHPs of six species, from 30 counties in four states, were tested, of which majority were collected from RJ. Howler monkeys  in RJ, a state recording three-decade history of autochthonous human cases of benign tertian malaria lacking epidemiological clarification of their origin. This is the first study to describe the infection rates by Plasmodium spp. in free-living NHPs in RJ, thereby matching the spatial distribution of P. simium in NHP with that of the local human cases of benign tertian malaria occurring due to this parasite. This study confirmed howler monkeys as the only reservoir of this zoonotic malarial parasite in RJ and reported that specific single nucleotide polymorphisms were present in all P. simium infected howler monkeys, despite their geographical origin of malarial foci. Moreover, this is the first study to record P. brasilianum/malariae in free-living NHPs from RJ, and to illustrate their widespread distribution in this state. Collectively, these findings help us in evaluating the simian malaria prevalence in the Atlantic Forests and in assessing the zoonotic characteristics of autochthonous human malaria in Rio de Janeiro, thus providing assistance in shaping surveillance and control.The present work comprises an unprecedented capture effort and large-scale field survey of  Plasmodium vivax-like parasites in the Atlantic Forest areas in Southeast Brazil, where no index case that could have introduced the parasite from a malaria endemic region, has been reported [P. vivax expected relapses, and recent visits to dense rain forest areas, where the bromeliad-inhabiting Anopheles mosquitoes belonging to the subgenus Kertezsia, specially An. cruzii, are found [An. cruzii is the main vector of “bromeliad malaria”, which is endemic in South and Southeast Brazil, and is the only known natural vector of simian malaria in the country [P. simium Fonseca [An. cruzii. The description of vertical movement of An. cruzii between the canopy and ground level in the Atlantic rain forest of Southern Brazil reinforced Deane’s hypothesis that part of the transmission of bromeliad malaria in Southern and Southeastern Brazil would be of zoonotic character, with monkeys being the parasite reservoir [In Brazil, more than 99% of malarial infections are acquired from the Amazon, and few isolated cases are occasionally found and recorded in regions outside the Amazon . Malariareported ,2. Thesere found . An. cru country . This pa Fonseca  in São P Fonseca . The pateservoir –8.Plasmodium have been described in the Neotropical non-human primates (NHP): P. brasilianum Gonder e Berenberg-Gossler (1908) and P. simium, almost indistinguishable from the human malaria parasites P. malariae and P. vivax, respectively [P. malariae and P. brasilianum, and P. vivax and P. simium [P. brasilianum is widely distributed compared to P. simium, with its presence from México to Southern Brazil, thereby infecting at least 11 genera of the five families of Neotropical primates  [P. simium has been found essentially in species belonging to two genera  [Two species of ectively ,6,9. Besectively ,5, molec. simium ,10,11. Peciidae) –15. In cPlasmodium sp. in the transmission foci as well as to determine the parasite reservoirs. Studies on the prevalence of P. simium and P. brasilianum infections in NHPs and their potential reservoirs in Southeast Brazilian states were conducted during 1960–1990s. Almost 800 NHPs were sampled and their blood slides were examined by microscopy, which is a less sensitive technique compared to the molecular assays, thus recording a variation in the Plasmodium infection from 10.9% in the states of Espírito Santo to 56.5% in SP [Alouatta, Brachyteles, Callicebus, Callithrix, Leontophitecus, and Sapajus) [An. cruzii being the most common anopheline mosquito [P. simium parasite [To completely understand the epidemiology of recent autochthonous malaria in Southeast Brazil, it is necessary to confirm the circulation of NHP 5% in SP . However5% in SP . InteresSapajus) , with Anmosquito . Consequmosquito . In respmosquito ,4,19. Moparasite ; howeverparasite . This stThe work was carried out between May 2015 and January 2019, with a total of 120 days of fieldwork at 44 sites in 30 counties of the Atlantic Forest biome, mainly in RJ but also in its bordering areas including the states of Minas Gerais (MG), ES, and SP. In this survey, we included forest fragments from lowlands areas to mountain valleys and escarpments of mountain chains such as Serra do Mar, which divides the state territory into two sides, one facing the ocean  and other the continent  . The choCallithrix, Leontopithecus, and Sapajus genera [Alouatta and Brachyteles genera, as well as for one titi monkey  [The expeditions included up to 10-day surveys in the forests, conducted by 2–6 trained people in the target areas to search NHPs. The capture method was selected according to the NHP species, behavior, and size . Brieflys genera ,23. The licebus) ,24. Sicklicebus)  were caplicebus) . A sampllicebus) .Plasmodium genus-specific gene [P. vivax and ssrRNA P. malariae and P. falciparum genes, as previously described [Plasmodium genus, P vivax, P. malariae, and P. falciparum assays, respectively [Giemsa’s solution stained thick and thin blood films were examined under a microscope with a 100× oil-immersion objective by two trained and independent microscopists. DNA was extracted from the blood clots as previously described  and fromfic gene ,29, and escribed . The senectively –30. MoreP. vivax-positive samples were subjected to P. simium differential diagnosis based on a mitochondrial SNP, the only genetic marker available to differentiate them [coxI gene fragment and subsequent enzymatic digestion, using primers and a previously described protocol, which detected 3.12 parasites/μL [ate them ,10. The Spleen fragments of a subsample comprising 16 howler monkeys (12 from RJ and 4 from ES), presumably found dead due to yellow fever, were fixed in Carson’s formalin-Millonig  and furtThe collection methods, biosafety, and anesthesia protocols adhered to the Brazilian law  on the use of animals in scientific research, and complied with the rules and regulations of Brazilian Ministry of Health , having In total, we examined 146 animals belonging to six species from 30 counties in four Brazilian states, with majority of animals being from RJ ; of thesPlasmodium was the howler monkey Alouatta guaribaclamitans . Trophozoites were the most commonly visualized blood forms; however, schizonts and gametocytes were also detected  were infected by P. simium, the causative agent of the autochthonous human malaria in this state , of which two were co-infected with P. simium  howler monkeys were infected with malarial parasites during sampling and, among these, seven 6.1% howlt decade . Six A. Previous malaria infections could be investigated by searching the malarial pigment in a subsample of 16 dead howler monkeys. Accordingly, the malarial pigment  was deteP. simium were diagnosed in howlers from four (66.6%) of the surveyed counties reporting human cases of benign tertian malaria in the state, and in a neighboring county (Sumidouro) where the human cases were never detected  in the 6 Kb region of the mitochondrial genome [P. simium specific SNP is carried by all parasites isolated from NHPs in the Atlantic Forest, which is in accordance with the study by Alvarenga et al. (2018) [P. simium was detected in howler monkeys captured in five out of 11 counties recently reporting autochthonous human malaria cases in RJ , four out of six (66.6%) howler monkeys were exposed to Plasmodium sp., when considering those with present and past malarial infections. This finding may also suggest that spontaneous healing from malarial infections may occur in the howler monkeys found in nature, as observed in the P. simium experimental infections in certain Neotropical NHPs [hologies , immune hologies , and sevhologies ,36, for hologies , was unahologies ,13. The l genome . Excludi. (2018) . Furtheres in RJ . Despiteensities ,37. Nevet Brazil ,5,38,39.n Brazil . Besidesnfection  may makecal NHPs  and in aP. simium infection in free-living howler monkeys in RJ (16.6%) was higher than that previously found in the bordering state of SP (5.8%) [A. g. clamitans was also the only free-living NHP from RJ in which blood forms by microscopy and plasmodial DNA were detected. Recently, DNA but not blood forms of P. simium was detected in captive Cebus and Sapajus from the Southeast [P. brasilianum DNA was detected in captive capuchin, titi, howler, and owl monkeys, as well as in tamarins and marmosets [P. simium near CPRJ [Plasmodium infections or only bear a transient parasitemia. Nevertheless, it is important to continuously monitor their potential role as a zoonotic Plasmodium reservoir, besides howler monkeys.The frequency of P 5.8%) ; however.8% ; howP (5.8%) . No prevoutheast . The paroutheast . P. brasarmosets ,27, mostP. brasilianum has been found in several NHP genera [Plasmodium [Plasmodium negative [P. simium infected blood, whereas splenectomized marmoset endured low parasitemia [Although P genera ,44–48 arasmodium . Similarnegative . Splenecasitemia . Thus, tP. brasilianum/malariae was found in a frequency similar to that of P. simium, six (14.3%) versus seven (16.7%), respectively–in howler monkeys from RJ, and mixed infections were recorded in two (4.6%) animals. P. brasilianum/malariae was the only malarial parasite detected in A. clamitans from ES  of Brazil, French Guiana, and Venezuela suggested infection of this simian quartan malaria parasite in humans [P. brasilianum/malariae in humans would be, therefore, expected to occur in RJ and other southeast states where P. simium has been described. In particular, the natural vector of both parasites is the same (An. cruzii) [P. brasilianum/malariae have not been consistently reported in the state. Further, strengthening of malaria surveillance either in residents or visitors of the Atlantic forest to evaluate the zoonotic potential of P. brasilianum/malariae in South and Southeast Brazil is recommended [ from ES . Besides from ES . Interesium, six 4.3% versseven 16.%, respec decades ,49,50. I in 2011 , and a sc forest . Similarc forest –54. Noteium, six .3% versun humans –57. InfeP. simium and P. brasilianum/malariae-infected howler monkeys  were from the forest coastal slope of Serra do Mar, where all autochthonous human malaria cases have been acquired [An. cruzii survival rates, supporting the sporogonic cycle of the Plasmodium. Sea moisture also favors the density of epiphyte shade bromeliads, the larval habitat of An. cruzii, and generates higher rainfall indexes [Plasmodium [Plasmodium transmission dynamics [An. cruzii, and consequently, define the occurrence or absence of simian malaria in nearby sites. Moreover, two genetic lineages of An. cruzii with partial reproductive isolation have been recently described in Serra do Mar, one curiously occurring in the coastal region and another in the continental slopes [Noteworthy, most of the acquired ,19. At lacquired ,59 may i indexes ,59, whicasmodium ,61. Vectdynamics . Indeed,l slopes . Comparel slopes ,19. CollPlasmodium infective to humans, it is plausible to suppose that dynamics of zoonotic transmission of malaria will undergo short or mid-term changes in RJ and bordering states affected by the YFV epizooties. In this context, we postulate that the rapid decrease of Alouatta populations would also reduce the source of plasmodial infection to An. cruzii, which would further hamper the circulation of Plasmodium sp. in the Atlantic forest. Despite the short duration since the 2016–2018 YFV epizootics, records from the Brazilian Ministry of Health surveillance program seem to confirm this scenario. In fact, there was an abrupt drop in the human malaria case records between 2018 and 2019 (only one autochthonous case) [Alouatta deaths, leading to plasmodial sources reduction, the role of howler monkeys for the occurrence of malaria in the Atlantic Forest would be reinforced.During the present study, a YFV outbreak erupted in the Southeast Brazil, a region without records of this virus presence for almost 80 years ,64. Hundus case) , which wAn. cruzii carrying esporozoites of P. simium acquired from howler monkeys could be a source of infection to An. cruzii or any other malarial vector present in the region. It is known that P. simium infected humans usually display scanty to null parasitemia, and can be cured spontaneously in few days without treatment and any relapse; moreover, the molecular detection of parasites during treatment follow-ups has been described [P. simium enzootic forest, and no secondary transmission directly derived from a human infected in the zoonotic cycle has ever been detected outside the sylvatic foci. These epidemiological and parasitological profiles appear to indicate that humans are not a source of P. simium infection for mosquitoes. Thus, determining vector competence of An. cruzii and other traditional human malaria vector occurring in the Southeast region  for transmitting P. simium and P. brasilianum between humans and from NHPs and humans and vice-versa is imperative.Previous sampling efforts on examining free-living NHPs in the Southeastern Atlantic Forest over the last 30 years revealed limited geographical coverage, with samplings essentially limited to the wildlife rescues or carried out in areas close to cities, or were based on few individuals ,38,39. Aescribed ,5. BesidS1 Table(DOCX)Click here for additional data file.


Plasmodium vivax was traditionally thought to be benign; however, nowadays it presents with a myriad of systemic complications like cerebral malaria, acute respiratory distress syndrome, acute kidney injury, acute pancreatitis, hepatic dysfunction, and disseminated intravascular coagulation, which were earlier attributed only to Plasmodium falciparum malaria. Here we report a case of a middle-aged man who presented with disseminated intravascular coagulation manifesting as symmetrical peripheral gangrene. What makes this case more interesting is that the malaria isolated was Plasmodium vivax instead of Plasmodium falciparum. Such findings were previously reported, but this is the first case where the patient was managed conservatively with antimalarial drugs without the need for amputation, which focuses on the very important role of early diagnosis and timely management.Plasmodium vivax malaria was found leading to disseminated intravascular coagulation causing this complication. He was started on artesunate and lumefantrine combination therapy and he recovered completely without the requirement of amputation.A 44-year-old Indian man from north India presented with history of fever of 2 days’ duration with severely painful cold extremities. No pulse could be recorded on examination. A diagnosis of symmetrical peripheral gangrene was made. During the etiological evaluation, Plasmodium vivax and its emerging complications. Also it correlates symmetrical peripheral gangrene with Plasmodium vivax malaria. It also emphasizes the importance of timely diagnosis and intervention to reduce mortality and morbidity.This case highlights the non-benign nature of  Anopheles mosquitoes. It is transmitted in 91 countries containing 3 billion people and causes approximately 1200 deaths each day [Plasmodium cause nearly all malarial infections in humans. These are Plasmodium falciparum, Plasmodium vivax, two morphologically identical sympatric species of Plasmodium ovale, Plasmodium malariae, and Plasmodium knowlesi [P. falciparum and P. vivax [Malaria is a protozoan disease transmitted by the bite of infected P. falciparum and mixed malarial infections [Symmetrical peripheral gangrene (SPG) is characterized by distal ischemic damage in two or more extremities, without large vessel obstruction. This syndrome has been reported in several conditions such as infections, disseminated intravascular coagulation (DIC), and low cardiac output states; it is rarely associated with fections , 4.P. vivax malaria presenting with DIC manifesting as SPG. This case is unusual because DIC and SPG have been reported extremely rarely in the literature in association with isolated P. vivax malaria [We report a case of isolated  malaria –7. AmputA 44-year-old Indian man, resident of New Delhi, India, a known alcoholic, presented with a 2 day history of fever. His fever was 39.44 °C (103 °F), continuous and was associated with chills and rigors. Two days after onset of fever, he developed painful bluish discoloration of both hands and feet. There was no history of bluish discoloration of tongue, breathlessness, or chest pain. There was no history of hypertension, diabetes, malaria, or any chronic illness in this patient.On clinical examination he was conscious, oriented, and well hydrated. His pulse was 80 beats per minute. Bilateral radial and dorsalis pedis arteries were feeble with bluish discoloration of fingers Fig.  and toesA provisional diagnosis of symmetrical arterial ischemia/obstruction with impending gangrene was made and he was investigated further.3/mm3 (4–11 × 103/mm3), and platelet count 8000/mm3 . Renal and liver functions were deranged with blood urea 90 mg/dL (15–45 mg/dL), serum creatinine 2.6 mg/dL (0.5–1.5 mg/dL), serum bilirubin 12.70 mg/dL (0.2–1.2 mg/dL), direct bilirubin 9.86 mg/dL (0.0–0.2 mg/dL), indirect bilirubin 2.84 mg/dL (0.0–1.0 mg/dL), albumin 2.4 g/dL (3.4–5.0 g/dL), aspartate transaminase 116 IU/L (5–40 IU/L), alanine transaminase 46 IU/L (5–40 IU/L), and alkaline phosphatase 116 IU/L (45–116 IU/L). His prothrombin time was 27.3 ; his international normalized ratio (INR) was 2.30. His random blood sugar was 64 mg/dL and arterial blood gas analysis was unremarkable.His hemoglobin was 15.9 g/dL (13–18 g/dL), hematocrit 44.8% (40–50%), total leukocyte count 3.9 × 10Arterial Doppler of both upper and lower limbs was normal and did not reveal any thrombus in any of the arteries. Radiography of his chest was normal.P. vivax  ruling out a mixed infection with P. falciparum. NS1 antigen and dengue serology were negative. Fibrin degradation products was > 20 μg/ml (< 5). His D-dimer level was 26.52 μg/mL (< 0.5) fibrinogen-equivalent units. Tests for antinuclear antibodies and antineutrophil cytoplasmic antibodies were negative.A peripheral smear (using Giemsa stain) for malaria revealed ring forms and schizonts of vax Fig. a, b. TheHe was treated with injection artesunate 120 mg followed by lumefantrine 480 mg and artemether 80 mg two times per day for 3 days followed by primaquine 15 mg (0.25 mg/kg) per day for 14 days; his glucose-6-phosphate dehydrogenase (G6PD) levels were normal. He also received six units of platelet concentrates, along with cautious use of intravenously administered fluids.The response to this line of treatment was striking and gratifying. He became afebrile by the second day of admission and by the third day all peripheral pulses were well felt along with normalization of blood pressure in all his limbs. Bluish discoloration of both hands and feet started fading by the third day and pain disappeared in the involved regions. His laboratory investigations normalized by sixth day of admission following treatment except for slightly raised bilirubin. He was discharged and no clinical or laboratory abnormality was reported on follow-ups at 2 weeks and 6 weeks.P. falciparum causes the majority of severe and fatal malaria cases and has overshadowed the public health importance of P. vivax malaria.P. vivax infection quite frequently [P. vivax is less pathogenic than P. falciparum in otherwise healthy patients, but can cause complicated and severe disease.Complications in severe malaria are either sequestration related, such as cerebral malaria, renal dysfunction, hepatic dysfunction, and acute respiratory distress syndrome (ARDS), or non-sequestration related, such as anemia and thrombocytopenia . Non-seqequently . P. vivaSPG is a well-documented but rare clinical syndrome. It is characterized by symmetrical distal ischemic damage leading to gangrene of two or more sites in the absence of large vessel obstruction or vasculitis. DIC is considered to be the final common pathway for the pathogenesis of SPG, irrespective of the etiology. It is proposed to be a cutaneous marker of the same .DIC is associated with a number of clinical conditions; generally involving activation of systemic inflammation . The comP. falciparum [Severe malaria has been associated with clinically apparent bleeding or DIC , 12. Coalciparum .P. vivax malaria by thromboelastography as well as the conventional coagulation tests [P. vivax malaria infection. Arora et al. [P. vivax infection complicated by SPG involving bilateral hands and feet. Two other cases [In a recent study, non-overt DIC was identified in more than 70% of patients of severe on tests . Howevera et al.  reporteder cases , 7 were P. vivax have been from India [Intriguingly all case reports of SPG in om India –7. WhethP. vivax malaria. It also emphasizes the need for a high index of suspicion of this clinical entity to differentiate it from conditions such as collagen vascular diseases, thrombotic thrombocytopenic purpura, and other conditions associated with peripheral gangrene. Early recognition and institution of appropriate treatment can prevent progression of gangrene and consequent requirement of amputation.The present case highlights SPG as a rare complication of


The World Health Organization (WHO) recommends testing of suspected malaria cases before treatment. Malaria rapid diagnostic test (mRDT) has been recommended for this purpose in endemic countries where microscopy is not accessible. However, its diagnostic performance remains a concern in clinical settings. We assessed diagnostic performance of RDT among febrile under-five children (U5) presenting at Oni Memorial Children's Hospital, Ibadan (OMCH).TM histidine rich protein II (HRP-2) RDT and microscopy. The sensitivity, specificity, positive and negative predictive values , false positive (FP), invalid rates (IR), likelihood ratio of positive and negative tests (LRP and LRN), were calculated.A cross-sectional study was conducted among 370 febrile U5 attending OMCH February to May, 2016. We examined their finger prick blood samples for malaria parasitaemia using CareStartTM HRP-2 RDT was 94.6% : 84.2-98.6), specificity: 91.4% (CI: 87.6-94.2), PPV: 66.3% (CI: 54.7-76.2), NPV: 98.9% (CI: 96.8-99.7), FPR 6.5%, IR 8.1%, LRP:10.6 and LRN:0.1.Mean age of the children was 28.17 ± 15.59 months. Malaria prevalence was 21.6% and 15.1% by mRDT and microscopy, respectively. Sensitivity of CareStartTM used in the study met the ≥ 95% sensitivity at 100 parasites/µL recommended by WHO. This finding provides clinical evidence that testing before anti-malarial treatment as recommended by WHO will identify cases of malaria infection and reduce unnecessary use of drugs. Healthcare workers should be educated on diagnostic accuracy of mRDT and adhere to the WHO's test-treat strategy for anti-malaria therapy.Diagnostic performance of CareStart Malaria is a life threatening tropical disease responsible for significant morbidity and mortality especially, among children and pregnant women, with 48 to 304 million new cases of malaria infections reported annually worldwide. Nigeria accounted for up to 29% of the total estimated malaria cases and 26% of deaths in the African region . AccuratTM malaria an HRP2-based immunochromatographic test which detects HRP2 antigen that is specific for Plasmodium falciparum has a very high performance level as reported by the most recent mRDT evaluation programme [TM RDT despite availability at a secondary level hospital, Oni Memorial Children's Hospital Ibadan (OMCH) could largely be as a result of varying reports of sensitivity in Nigeria. We therefore assessed the diagnostic performance of CareStartTM RDT among febrile under-five children (U5) who presented at OMCH.This necessitated the introduction of mRDT kits in the early 1990s with the potential to overcome the weaknesses associated with microscopy . Malariarogramme  and is arogramme , howeverrogramme , 16. Somrogramme , 16. Thirogramme , 47.0% irogramme , 40.3% irogramme . The levStudy area: A hospital-based cross-sectional study carried out at Oni Memorial Children's Hospital, Ibadan in Ibadan South West Local Government area, Nigeria from February to May, 2016. Oni Memorial Children's Hospital was purposively selected because it is the only secondary level health facility dedicated to children below 10 years in Oyo State. The hospital is a 65-bedded capacity facility, consists of an emergency unit, intensive care, out-patient unit and 2 specialised units for Heamatology Day Care and Neurological cases. It serves as a referral center for all the health centers within and outside Ibadan. It also provides routine immunization and child welfare services. An average of 900 children of which 500 (55.6%) are under-five are attended to on a monthly basis, while about 750 (83%) of these children are presumptively diagnosed as malaria cases thereby necessitating laboratory confirmation. Though, CareStartTM RDT was available at the OMCH, microscopy remained the mainstay of diagnosing malaria.Sample size estimation: The number of participants for the study was calculated using the following formula:α = standard normal deviate corresponding to 2-sided level of significance at 5% = 1.96; p = prevalence of malaria parasitaemia among under-five children presenting with malarial at a general hospital in Ota, Ogun State, 70% [ where: nate, 70% . q = 1 -2 x 0.70 x 0.30/(0.05)2 = 323 Adjusting for non-response rate (10%) calculated using the following formula:(1.96)= n x 1/(1-r) Where: n = sample size calculated; r = non response rate (10%); = 323 x 1/(1-0.1) = 359. The minimum sample size for the study was 370.Recruitment of participants and data collection: A total of 370 febrile under-five children who presented at the emergency and general out-patient (GOP) units of the OMCH with history of fever (axillary temperature > 37.5°C) and a provisional diagnosis of malaria and whose parent/caregivers gave informed consent for the study were recruited consecutively over a period of two months. We excluded all children who were severely ill and because we could not analyze the drug level of those who have taken any anti-malaria drug, they were also excluded. Data on demographic characteristics, child characteristics and episodes of malaria illness were collected from the caregivers using pre-tested interviewer administered questionnaire.Laboratory examination: Capillary blood sample of each febrile child obtained by finger prick was collected on frosted end slides and CareStartTM Histidine-rich protein 2 (HRP-2) RDT Cassette. Thick and thin blood films were prepared, stained with Giemsa same day and examined microscopically according to WHO standards [-1 = parasite count/number of white blood cells counted × 8000 Microscopic examination of all stained slides was conducted by a trained WHO certified malaria microscopist with 7 years of experience, following standard methods [TM malaria Pf RDT, Lot No. M015C01 manufactured in March, 2015 by Access Bio Incorporation, New Jersey, USA. This was obtained directly from the pharmacy Unit of OMCH through United State agency for International Development (USAID) representative. All the participants were tested by adhering strictly to manufacturer's guidelines.tandards . Parasit methods . Each saQuality control: The positive control check of the RDT was done with positive (dilutions at 200 and 2000 parasite/µL of blood) and negative (zero parasitaemia) confirmed by microscopy.Data analysis: Data was analyzed using Statistical Package for Social Sciences (SPSS) software version 16.0. Data were summarized using mean, frequencies, proportions and 95% confidence interval (CI). Parameters for mRDT diagnostic performance was calculated using the standard WHO format as follows: (a) True positive (TP) = sample is positive by both microscopy and CareStartTM HRP-2; (b) True negative (TN) = sample is negative by both microscopy and CareStartTM HRP-2; (c) False positive (FP) = sample is positive by CareStartTM HRP-2 but negative by microscopy; (d) False negative (FN) = sample is negative by CareStartTM HRP-2 but positive by microscopy.Ethical consideration: Ethical approval for the study was obtained from Oyo State Ethical review committee . Informed consent was also obtained from the caregivers.TM HRP-2 detected 53 true positives (TP), 27 false positives (FP), 287 true negatives (TN) and 3 false negatives (FN). The microscopy detected 56 malaria positive cases among the participants, 55 Plasmodium falciparum and 1 Plasmodium malariae, while the RDT detect 80 positive cases of Plasmodium falciparum (TM HRP-2 RDT was 94.6% (95% CI: 84.2-98.6) and specificity of 91.4% (CI: 87.6-94.2) . The male participants constitute 211 (57.0%), other children characteristics are shown in lciparum . The Sen.6-94.2) .TM RDT HRP2 Pf shows a good performance with high sensitivity of 94.6% and comparable to the 94.3% and 93.0% sensitivity reported in Ibadan by Falade et al [Despite several interventions, malaria still continues to be a major public health challenge especially among under five children. Malaria parasitaemia prevalence of 15.1% and 21.6% was observed with light microscopy and mRDT respectively in this study. This observed prevalence is lower than previously reported prevalence among children presenting at health facilities in Nigeria which ranged between 27.7% and 84.7% in studies by Ikeh and Teclaire , (56.9%)de et al  and in Sde et al  respectide et al , Jos by de et al , Lagos bde et al , Enugu bde et al , and Zamde et al , respectTM RDT HRP2 Pf, while the current study gave a sensitivity of 94.6%, Sheyin and Bigwan, and Abdulkadri reported 78.4% and 40.3% respectively. These observed conflicting reports may be due to the functionality of RDTs which remains unable to detect parasites at low densities (< 200-400/ µL). However, other factors such as storage which were not considered in the current study may be responsible for the differences which has eroded the confidence of end users. Although the RDT specificity was high (91.4%), 27 children still had false positive results while microscopy was negative. This may imply that the children did not have active malaria, or had incomplete treatment with antimalarial drug, however, the positive RDT result could be attributed to delayed clearance of antigen from the circulation. It has been shown that malaria antigen clearance could be delayed for up to 4 weeks even, when treatment was successful Abdulkadri et al [TM also support the validity on its use in excluding/eliminating malaria as the cause of febrile illness and allows early decision making in looking out for other causes of febrile illness in the under-fives, which will enhance their survival, thereby reducing mortality in under-fives. The other findings which include invalid rate of 8.1% are within the acceptable rates according to WHO assessment of RDTs 2014-2015 round six [The disparity in reported sensitivity may not be because the studies used different kits, for instance while Ajumobi, Falade, and Jeremiah all used SD Bioline malaria Pf RDT, they still reported discordant sensitivity of 100%, 94.3% and 47% respectively. Also for the CareStartri et al . High spound six .TM HRP-2 RDT met the ≥ 95% sensitivity at 100 parasites/µL recommended by WHO. The study provides the clinical evidence to justify its use for testing before treatment as recommended by WHO. Use of RDT should be encouraged for diagnosis of malaria using a protocol that encourage further evaluation of febrile children with negative RDT results using microscopy. Efforts should be made to educate healthcare workers on the importance of RDT test kits and enforcement of protocols that supports the WHO's test-treat strategy for anti-malaria therapy. There should be regular communication of availability of mRDTs among all stakeholders in every health care facilities and also Government at both federal, state and local levels should limit the number of mRDT product types for the country based on local data on performance. Also the varying sensitivities in different parts of Nigeria calls for more study to unravel the circumstances affecting the performance of the rapid test kits.The diagnostic performance of CareStartLimitations of the study: Report on the use of antimalarial drug before presentation cannot be verified but we elicited this from the caregivers as much as possible. The negative results could not be confirmed by polymerase chain reaction (PCR) because of limited funds.Accurate diagnosis of malaria remains a challenge in Nigeria and empirical treatment of fevers, with antimalarial medicines continues in resource-poor settings;Diagnosis of malaria based on blood film microscopy using thick and thin smears stained with Giemsa has remained the gold standard for many years;Nigeria adopted mRDT as a diagnostic tool, where microscopic diagnosis is not feasible but there have been varying reports of sensitivity of different types of mRDT kits ranging from 8.0 % to 94.3% in Nigeria.TM HRP-2 RDT met the ≥ 95% sensitivity at 100 parasites/µL recommended by WHO;Diagnostic performance of CareStartThe study provides the clinical evidence to justify its use for testing before treatment as recommended by WHO;Government at both federal, state and local levels should conduct lot testing of batches of mRDT on a regular basis to ensure the mRDT in use at health facilities are of high diagnostic performance.The authors declare no competing interest.


Plasmodium falciparum MSP-119 antigen in children from five contrasting bioecological zones in Cameroon.Studies reporting the natural immune responses against malaria in children from different geographical settings in endemic areas are not readily available. This study was aimed at comparing the immune responses against P. falciparum MSP-119 antigen using standard ELISA technique.In a cross-sectional survey, children between 2 and 15 years, were enrolled from five ecological strata including the south Cameroonian equatorial forest, sudano-sahelian, high inland plateau, high western plateau, and the coastal strata. The children were screened for clinical malaria . Their antibody responses were measured against In all, 415 children comprising 217 (52.3%) males participated. Total IgG and IgG1–IgG4 titres were observed to increase with age in all the strata except in the sudano-sahelian and high inland plateau strata. Total IgG and IgG1–IgG4 titres were significantly higher in the coastal strata and lowest in the high inland plateau (for IgG1 and IgG2) and sudano-sahelian strata (for IgG3 and IgG4). Titres of the cytophilic antibodies (IgG1 and IgG3) were significantly higher than the non-cytophilic antibodies (IgG2 and IgG4) in all the strata except in the sudano-sahelian and high inland plateau strata. Total IgG and IgG subclass titres were significantly higher in children positive for clinical malaria compared to negative children in all study sites except in the high western plateau and coastal (for IgG1 and IgG3), and the sudano-sahelian strata . Furthermore, a significant positive correlation was observed between parasite density and IgG2 or IgG4 titres in all study sites except in the south Cameroonian equatorial forest and sudano-sahelian strata.19 vary considerably in children from the different bioecological strata in Cameroon and could be linked to the differential exposure to malaria in the different strata. Furthermore, the rate of antibody acquisition was not observed to increase in an age-dependent manner in low transmission settings.This study showed that antibody responses against MSP-1The online version of this article (10.1186/s12936-019-2654-9) contains supplementary material, which is available to authorized users. Plasmodium, remains a major public health challenge in sub-Saharan Africa (SSA). In 2017, there were an estimated 219 million cases and 435,000 deaths attributed to malaria worldwide [Malaria, a disease caused by a parasitic protozoan of the genus Plasmodium falciparum is the predominant species causing malaria in Cameroon [Malaria in Cameroon is a major cause of morbidity and mortality especially in children . In the Cameroon  and the Cameroon .The epidemiology of malaria in Cameroon is complex and has been described as “Africa in miniature” , becauseIn malaria, immunoglobulin G (IgG) antibodies are known to play a vital role in combating infection by reducing parasitaemia and clinical symptoms –13. AmonPlasmodium spp. It is the most abundant merozoite surface protein and is thought to be involved in the initial attachment of the merozoite to the erythrocyte surface [19) has been recognized as the target of IgG-based protective immunity [19 was selected over the other MSP-1 molecules because of the fine specificity of MSP-119 specific antibodies [19 specific antibodies are potent inducers of neutrophil antibody-dependent respiratory burst (ADRB), which correlates with acquired clinical protection. Furthermore, studies have demonstrated the role of the cytophilic antibodies in ADRB [This study evaluated the humoral (IgG) immune responses against merozoite surface protein 1 (MSP-1). MSP-1 is one of the best-characterized proteins in  surface . The 19 immunity  and is aimmunity . MSP-119tibodies , 27 couptibodies  showed t in ADRB , 30.19 recombinant antigens were compared. The aim was to generate data that will improve understanding of the immune response against malaria in different geographical zones and may be useful in designing future trials for testing of malaria vaccine candidates.Previous studies have shown that malaria transmission in Cameroon decreases steadily northward, from the coastal (C) strata in the south to the SS strata in the north , 6, whilFive out of the six bioecological strata of malaria were randomly selected and participants were enrolled from five study sites, one for each strata including: Maroua in the sudano-sahelian strata, Ngaoundere in the high inland plateau strata, Yaoundé in the south Cameroonian equatorial forest strata, Bamenda in the high western plateau strata and Limbe in the coastal strata Fig. .Fig. 1MaPlasmodium falciparum is the main species causing malaria and its main vector is Anopheles gambiae [The sudano-sahelian (SS) strata constitute the dry savannah zones and the steppes to the north of the country. It lies between latitudes 13° N and 8° N. The SS strata is characterized by a hot semi-arid climate and the vegetation is of the sahelian type. There is not much rainfall in this zone; the mean annual rainfall is 794 mm (ranging from 0 to 245 mm). The average temperature is 35 °C (ranging from 28 to 45 °C). Malaria in the SS strata is unstable with a risk of an epidemic and severe clinical forms that affect all ages [ gambiae .P. falciparum. Data on the entomological inoculation rate was not readily available for Ngaoundere, where this study was conducted.The high inland plateau (HIP) strata is found in the very heart of Cameroon, between latitudes 8° N and 6° N. It is characterized by the Guinea-Savannah type vegetation and the Sudano-Guinean tropical climate is tempered by the altitude (1100 m on average). The average temperature is 22.1 °C and mean annual rainfall is 1485 mm (ranging from 0 to 280 mm). Malaria in the HIP strata is tropical and stable with seasonal outbreaks, caused mainly by P. falciparum and An. gambiae is the principal vector. In Yaoundé, where this study was conducted, the entomological inoculation rates is reported as 6 bites per man per month during the rainy season [The south Cameroonian equatorial forest (SCEF) strata lie between latitudes 5° N and 2° N. It is characterized by the Guinea-type equatorial climate and the vegetation is comprised of the equatorial evergreen forest. The average temperature is 23.7 °C (ranging between 22.6 °C and 24.6 °C) and the mean annual rainfall is 1643 mm. Malaria in the SCEF strata is holoendemic, caused mainly by y season .P. falciparum and An. gambiae is the main vector [The coastal (C) strata corresponds to the only coastal region in Cameroon. It ranges from Campo to Mamfe. Its altitude is inferior to 300 mm and this is a vegetable cul-de-sac often swallowed up by the monsoon. The climate is warm and temperate. The average temperature is 19.9 °C (ranging from 15.8 to 32.8 °C) and the mean annual rainfall is 1174 mm (ranging from 27 to 617 mm). In this zone, malaria transmission is highest in the country, caused mainly by n vector .P. falciparum is the main species and An. gambiae is the main vector. Data on the entomological inoculation rate was not readily available for Bamenda, where this study was conducted.The high western plateau (HWP) strata is a polygon-shaped region stretching 300 km by 200 km. It is characterized by the tropical savanna climate and Sudan savanna forms the dominant vegetation. The mean temperature is 21.5 °C (ranging from 20.1 to 23.0 °C) and the mean annual rainfall is 2145 mm (ranging from 9 to 383 mm). Malaria transmission in the HWP strata is stable, occurring all year long. This was a cross-sectional comparative survey conducted on children from five bioecological strata of malaria in Cameroon. Data were collected between April and July 2015 in all the strata (to coincide with the rainy season during which transmission is higher).Children (2–15 years) of both sexes were randomly selected from the community in the different study sites. Eligible participants here were those who had not been on any anti-malarial drug for at least a week prior to enrolment.Using the effect size of 0.18 deduced from the study by Nebie et al. , and usiIn order to ensure that the data obtained is a true representation of the study site, a multistage sampling technique was used to sample apparently healthy children (2–15 years) from the community by firstly, randomly selecting three neighbourhoods, and secondly, randomly selecting 135 houses within the neighbourhoods and one child per house who met the inclusion criteria, was selected to obtain the required sample size. This was done in all the study sites.From every participant, about 3 ml of venous blood was collected into EDTA anticoagulated tube and non-coagulated (dry) tube following antiseptic techniques. Sample from the EDTA tube was used in the preparation of blood films and performing complete blood count (CBC), while sera from the dry tube were used to perform ELISA.The prepared blood films were air-dried and stained with 10% Giemsa (1 in 20 dilutions) for 25–30 min . The blo19 antigen (expressed in Escherichia coli and purified to 95%) using a standard ELISA technique as earlier described [19) at a final concentration of 1 µg/ml. The unbound antigens were removed by washing 3 times with 0.1% Tween/PBS washing buffer. After washing, 150 µl/well of 3% skimmed milk powder in Tween/PBS was used to block the wells for 8 h at 4 °C, followed by plate washing as described above. After the second wash, sera samples (diluted 1 in 100) were added in duplicate, along with positive control serum  and 10 negative control sera from non-exposed German adults (provided by Andreas Latz). The plates were then incubated at 37 °C for 1 h before washing as described above. Peroxidase-conjugated goat anti-human IgG  for IgG (1/40000) and mouse anti-human IgG1, IgG2, IgG3 and IgG4  for IgG1 (1/6000), IgG2 (1/4000), IgG3 (1/6000) and IgG4 (1/5000) were added (100 µl/well) and incubated for 30 min at room temperature (RT). Afterward, 100 µl/well TMB substrate was added and incubated for 15 min in the dark. The reaction was then stopped by adding 100 µl/well of 0.2 M sulphuric acid (H2SO4) and absorbance was read at 450 nm with an ELISA plate reader, BioTek® ELx800TM . A standard curve derived from serial dilution  of positive control sera for all test plates, was used to convert antibody responses to arbitrary units (AU), with the absorbance of the lowest dilution corresponding to 100 AU [The total IgG and IgG subclass responses were measured against PfMSP-1escribed . Brieflyo 100 AU .® version 12.1 software  and SPSS statistical software version 20 .Frequency tables and charts were used to present data. Age was grouped into three categories: 1 (< 5 years), 2 (5–9 years), 3 (≥ 10 years). The statistical tests performed included the Student’s t-test and ANOVA for the comparison of group mean, multiple linear regression analysis was also used to compare antibody concentrations between groups adjusting for possible confounding . The data was checked for clustering using the K-means cluster and the Hierarchical cluster methods and clustering was insignificant. All continuous variables were standardized prior to statistical analysis by logarithmic transformation. Statistical significance was set at p ≤ 0.05. For the comparison of IgG antibody responses, clinical malaria was defined as malaria parasitaemia (≥ 5000 parasites/µl) plus fever (axillary temperature ≥ 37.5 °C). Data collected were entered into an Excel spreadsheet and analysed using the Stata2 = 1.732, p = 0.785) and their mean ages (p = 0.439).In all, 415 children consented and took part in the study. There were 83, 82, 82, 85 and 83 participants from Bamenda, Limbe, Maroua, Ngaoundere, and Yaoundé, respectively . Malaria prevalence was highest in Limbe  and lowest in Maroua (in the sudano-sahelian strata) (p < 0.0001). Malaria prevalence was highest in children between 5 and 9 years and lowest in children below 5 years (p = 0.004). No significant difference was observed between malaria prevalence and gender (p = 0.227)  adjusting for gender and study site Fig. . Total I adjustinGlobally, total IgG levels were observed to be independent of gender (p = 0.055) adjusting for age and study site. Although not significant, the total IgG levels were higher in females in all the study sites except in Limbe where it was higher in males. Site-specific analysis of the levels of the different IgG subclasses revealed no significant association with gender, except in Maroua where females were observed to have significantly higher titres of IgG2 (p = 0.030) and IgG4 (p = 0.016) adjusting for age and study site.Total IgG levels were observed to vary with study site, being highest in Limbe and lowest in Ngaoundere Fig. . MultiplSite-specific analysis of the different IgG subclasses revealed that the cytophilic antibodies (IgG1 and IgG3) were consistently higher than the non-cytophilic antibodies (IgG2 and IgG4) in all the study sites except in Maroua and Ngaoundere , Limbe  and Yaoundé , but not in Ngaoundere (in the high inland plateau strata) and Maroua (in the sudano-sahelian strata). In Ngaoundere, a generally flattened trend was observed for all the IgG subclasses while in Maroua, total IgG, IgG3, and IgG4 did not increase with age as well. This is contrary to numerous reports that immunity to malaria increases in an age-dependent manner [However, the increase in antibody response with age was not observed in all study sites. Total IgG and IgG1–4 titres against MSP-1t manner , 46. TheThe immune responses against malaria were also observed to be significantly associated with the geographical area. Total IgG and IgG1–4 antibody titres were highest in Limbe and decreased steadily northward to Ngaoundere and Maroua. Total IgG, IgG1 and IgG2 were lowest in Ngaoundere meanwhile IgG3 and IgG4 were lowest in Maroua. These discrepancies could be attributed to differences in the transmission intensity of malaria in the different ecological strata. Malaria transmission could be described as hyperendemic in Limbe, holoendemic in the Yaoundé, mesoendemic in Bamenda and Ngaoundere, and seasonal and hypoendemic in Maroua . The varThe cytophilic antibodies (IgG1 and IgG3) were consistently higher than the non-cytophilic antibodies (IgG2 and IgG4) in all the study sites except in Maroua and Ngaoundere. The findings of generally higher titres of IgG1 and IgG3 in this study is in conformity with other studies performed in Cameroon , 51 and 19). Studies using other recombinant antigens will be required to confirm this observation. However, site-specific analysis of the different IgG subclasses revealed no significant difference between children positive and negative for clinical malaria for IgG1 and IgG3 in Bamenda (in the HWP strata) and Limbe (in the C strata), and for all the IgG subclasses in Maroua (in the SS strata), suggesting that infection is required to mount an effective immune response against the malaria parasite. Furthermore, a significant positive correlation was observed between malaria parasite density and IgG2 or IgG4 in Bamenda, Ngaoundere and Limbe, suggestive of their role in the pathogenesis of malaria. The non-cytophilic antibodies especially IgG4 are known to be pathogenic, and their presence correlates with the severity of malaria [In this study, total IgG levels were higher in children that were positive for clinical malaria compared to negative children overall. This demonstrates that infection might lead to the boosting of antibody levels, resolved infection likely leads to reduced antibody levels due to decay. This is in conformity with other studies , 57. The malaria , 18, 19.19) was used to evaluate the antibody responses in the current study, which does not necessarily provide a complete picture of the immune responses against malaria in children in the different study sites. Further research using more recombinant antigens will be required in the study area to give a clearer picture. In addition, longitudinal studies will be required in the different bioecological strata to identify the antibodies which are protective against malaria.This study which clearly demonstrates the variation in the immune responses against malaria parasites in children from different geographical areas in Cameroon is however limited in that participants were enrolled only during the rainy season during which malaria transmission is generally higher. Studies enrolling children during the rainy and dry seasons will be required to give a clearer picture. Furthermore, only one recombinant antigen , and lowest in the high inland plateau and sudano-sahelian strata . The immune responses were observed to increase with age in all study sites except in the sudano-sahelian (where only IgG1 and IgG2 levels increased with age) and high inland plateau (where none of the antibodies levels increased with age) strata. Furthermore, the cytophilic antibodies (IgG1 and IgG3) were consistently higher than the non-cytophilic (IgG2 and IgG4) antibodies in all the study sites except in the sudano-sahelian and high inland plateau strata. No significant differences were observed in all the antibodies levels between children positive and negative for clinical malaria in the sudano-sahelian strata. These findings may have significant implications for the design of trials for testing of malaria vaccine candidates in the country. However further research is required to identify the protective antibodies against malaria in the different ecological strata.Additional file 1: Figure S1. Variation of total IgG, IgG1-4 antibody levels to MSP-119 with age in the different study site. The line shows the LOESS smoothed estimate of the geometric mean.Additional file 2: Figure S1. Plots of IgG subclass levels against malaria parasite density in Bamenda. These revealed significant positive correlations between IgG2 and IgG4 and parasite density in the study population. Figure S2. Plot of IgG subclass levels against malaria parasite density in Yaounde. These revealed no correlations between IgG subclasses and parasite density in the study population. Figure S3. Plot of IgG subclass levels against malaria parasite density in Ngaoundere. These revealed significant positive correlations between IgG1, IgG2 and IgG4 and parasite density in the study population. Figure S4. Plot of IgG subclass levels against malaria parasite density in Maroua. These revealed no significant positive correlations between IgG subclasses and parasite density in the study population. Figure S5. Plots of IgG subclass levels against malaria parasite density in Limbe. These revealed significant positive correlations between IgG4 and parasite density in the study population.