Malaria is a leading cause of imported febrile illnesses in pediatric travelers, but few studies have addressed severe imported pediatric malaria. We aimed to determine the risk factors and the features of imported pediatric severe malaria.falciparum malaria from January 1st 1996 to December 31th 2005.We conducted a retrospective, descriptive study using the French National Reference Center for Imported Malaria database, in children aged 0–15 years who were hospitalized with a falciparum malaria were compared to identify risk factors for severe cases. In the hospitals that reported more than five severe cases during the study period, we evaluated severe cases for prognostic factors and assessed the accuracy WHO criteria for predicting severity. Given the rarity of deaths, adverse outcomes were defined as requiring major therapeutic procedures (MTPs)—e.g., sedation, mechanical ventilation, nasal oxygen therapy, blood transfusions, hemodialysis, fluid resuscitation—or pediatric intensive care unit (PICU) admission.Uncomplicated and severe cases of falciparum malaria were: age <2 years  and a travel in the Sahelian region . Of 422 severe malaria cases, a stay in a Sahelian region, lack of chemoprophylaxis, age <2 years or thrombocytopenia <100 x 10^3/mm^3 predicted adverse outcomes. Except for the hyperparasitemia threshold of 4%, the main WHO 2000 criteria for severe malaria reliably predicted adverse outcomes. In our study, the threshold of parasitemia most predictive of a poor outcome was 8%.Of 4150 pediatric malaria cases included in the study, 3299 were uncomplicated and 851 (20.5%) were severe. Only one death was recorded during this period. Predictors for severe In imported pediatric malaria, children younger than 2 years deserve particular attention. The main WHO 2000 criteria for severity are accurate, except for the threshold of hyperparasitemia, which should be revised. According to the World Health Organization (WHO), malaria-related mortality has declined significantly for a decade. However in 2014 malaria was responsible for about 438,000 deaths worldwide, mostly children under 5 years of age living in sub-Saharan Africa .We conducted a retrospective study using data from the National Reference Centre of Imported Malaria , a national network of hospital correspondents who report their cases of imported malaria. The “CNR du paludisme” gives estimations of the annual incidence of imported malaria in mainland France [All cases reviewed for this study occurred between 1 January 1996 to 31 December 2005 in mainland France in children aged 0–15 years, and were extracted from the national database.First uncomplicated and severe pediatric malaria cases reported to the “CNR du paludisme” during this period were compared looking for predictors of severe forms.Then severe malaria cases (including those with uncomplicated hyperparasitemia) from hospitals that had reported more than 5 severe pediatric malaria cases during the study period were reviewed using a standardized data collection form. In these hospitals, additional cases of severe malaria, not declared to the CNR du paludisme, were found and were added to the cases issued from the national database . Epidemifalciparum malaria was made by the detection of asexual forms (trophozoites) of P. falciparum, alone or associated with other Plasmodium species, using the examination of thick and /or thin blood smears.The diagnosis of A severe case was defined by the presence of at least one severity criterion according to the 2000 WHO definition, including uncomplicated  hyperparasitemia ≥ 4%. The 4% threshold was used since almost all reported cases occurred among children born and living in France, and thus considered non-immune .Anemia was defined as haemoglobin (Hgb) <110 g/L and severe anemia as a Hgb <50 g/L or hematocrit <15%. Thrombocytopenia was defined as a blood platelet count <150 x 10^3/mm^3, but we considered a 100 x 10^3/mm^3 threshold for analysis of severity, according to data from non-immune children living in a hypoendemic area [Among severe cases of malaria, multiple organ dysfunction (MOD) were defined by the presence upon hospital admission of at least two of the following organ failure criteria: (i) central nervous system ; (ii) respiratory distress; (iii) renal failure, (iv) liver failure or digestive disorders ; (v) thrombocytopenia <150 x 10^3/mm^3 or spontaneous bleeding.falciparum malaria was contracted were located in West Africa, Central Africa, Southern and East Africa, and the Indian Ocean . These countries were classified according to epidemiological strata [The countries where l strata . This cll strata . We usedHaut Conseil de Santé Publique). During the period of the study, recommendations were adapted to the level of sensitivity of antimalarial drugs: zone 0 ; zone 1 (no chloroquine resistance): chloroquine; zone 2 : proguanil or atovaquone/proguanil; zone 3 : atovaquone/proguanil or mefloquine or doxycycline.The adequacy of chemoprophylaxis was assessed according to adherence to French recommendations, depending on the country visited and the season of exposure, which are published every year under the authority of the High Council of Public Health .The French recommendations for the treatment of children malaria during the period of study were halofantrine or mefloquine in uncomplicated malaria or quinine intravenously without loading dose in severe malaria  the need for at least one major therapeutic procedure (MTP) including sedation, mechanical ventilation, nasal oxygen therapy, blood transfusions, hemodialysis or fluid resuscitation, or (ii) an admission to a pediatric intensive care unit (PICU).Statistical analyses were performed using StataTM version 9 software.The comparison of variables required the use of a chi 2 test or Fisher exact test under the conditions of applications. The mean comparison was made from the Student's t tests.Variables included in the univariate and multivariate analyses were age, sex, geographical distribution, chemoprophylaxis and platelet count.p-value < 0,25 were selected for multivariate analysis. The variables were selected by a backward stepwise method (Hosmer and Lemeshow). Only significant variables at 5% were retained at the end of this analysis.The search for predictors of poor outcomes, defined by a requirement for a major therapeutic procedure or PICU admission, was performed using a logistic regression model. A first step was to select the variables  associated with the endpoint  in univariate analysis. Factors associated with a The criteria for severe malaria  were morversus <4%), variable in three classes  and continuous variable. Regarding the analysis of parasitemia as a continuous variable, a ROC curve (Receiver Operating Characteristic Curve) was drawn (representation of the sensitivity versus 1 minus the specificity) to determine the most accurate threshold associated with a poor outcome.Parasitemia was evaluated using several definitions: dichotomous criterion .As CNR’s data were anonymous, we could not obtain an informed consent from children or parents. Data collected in hospital files were also given anonymously, so we could not reach children or parents to obtain an informed consent. Finally, the study was designed in 2004–2005. In 2004, August, French law introduced the ‘Comité consultatif sur le traitement de l'information en matière de recherche dans le domaine de la santé’, as a national ethics committee for observational studies. That committee was not fully operational when we conducted this study. All the data were collected retrospectively and anonymized in a standardized case report form in the database.From 1996 to 2005, 4,150 cases of pediatric malaria were reported to the CNR du paludisme, of which 3,299 were uncomplicated and 851 were severe .Reports of imported malaria have tended to decrease since 1999. However, the annual ratio of severe to uncomplicated forms remained stable during the study period.The distribution of the epidemiological and biological characteristics of the patients is shown in Only 14 children (0.4%) had visited non-African regions , none with severe malaria. During their stay, 36% of children received no chemoprophylaxis. Only 6% of parents regularly gave their children prophylaxis consistent with French national recommendations , quinine (16.1%) or mefloquine (10.6%). Halofantrine was used most commonly but usage declined steadily from 1996 to 2005: 85% to 47% in uncomplicated cases, and 67% to 28% in severe cases, respectively. Intravenous quinine was used more often in severe cases (26.6%) than in uncomplicated forms (13.4%) (p = 0.0001) over the study period. Mefloquine was increasingly prescribed for both forms from 1996 to 2005: 3% to 25% in uncomplicated cases, and 2% to 33% in severe cases, respectively.All patients with uncomplicated malaria recovered uneventfully. Among the severe cases, 838 children (98.5%) had an uneventful course, 11 patients (1.3%) had transient clinical worsening, one (0.1%) had neurological sequels and one (0.1%) died.versus 25.1%, p = 0.002) and among children under two years .Comparison between uncomplicated and severe malaria was performed in univariate analysis. The proportion of severe cases was significantly higher among children travelling in a Sahelian country than in those returning from other countries . The mean rate of Hgb was significantly lower in severe than in uncomplicated malaria . The mean platelet count was also significantly lower in severe than in uncomplicated cases .The average parasitemia was significantly lower in uncomplicated cases than in severe cases   and a stay in Sahel . Conversely, mixed  = 0.02) .During the study period, 14 hospitals reported at least five cases of severe pediatric malaria, including 12 in the Region Île-de-France and 2 in the city of Marseilles, yielding 422 cases that were included in a detailed analysis of severe malaria cases .The annual incidence has generally decreased since 1999. The average patient age was 5.8 ± 4 years and the sex ratio (M/F) was 0.9. In our series, most of children (90.7%) lived in France. The most visited countries were the Ivory Coast (22%), the Comoros Islands (20%), Mali (16%), Senegal (13%) and Cameroon (9%). The average length of stay was 64 ± 97 days.Globally, 73% patients were reported (by themselves or parents) as taking chemoprophylaxis during their stay. Of these, 44 children (10.4%) were fully compliant with recommendations. The use of vector control measures  was commented on in 159 children, of whom 16% were reported sleeping under a bed net and 10% using repellents.The average time from the onset of symptoms to diagnosis malaria was significantly longer in uncomplicated than in severe malaria cases .Salmonella sp., Campylobacter jejuni, Shigella sp.) (n = 12), hepatitis A (n = 8), urinary tract infection (n = 4), ringworm (n = 4) and viral infection  (n = 3). No case of bacteremia was noted.Besides WHO criteria, clinical signs present on admission were: temperature ≥38.5°C (64%), hepatomegaly (26%), vomiting (25%), splenomegaly (23%) and headache (19%). Only 2 patients had pupillary abnormalities . Co-infection was diagnosed in 31 children: gastrointestinal infections  children, and <100 x 10^3/mm^3 in 185 (45%).Treatments were halofantrine , intravenous quinine , or mefloquine . No severe side effects were recorded.Of 422 patients, 100 (23.7%) required at least one MTP  and 43 (10%) stayed in a PICU. The distribution of numbers of units of transfusions received across all subjects was: 1 unit (N = 51), 2 units (N = 13), 3 units (N = 3), > 3 units (N = 2). Two children received massive red blood cell transfusions, i.e., 15 units each.The clinical course was uneventful in 366 children (86.7%). Thirty children (8.2%) had a relapse within 28 days of the first episode, usually after a single course of halofantrine (60% of relapses). All relapses were uncomplicated and patients fully recovered using oral antimalarial drugs. Finally, only one child died and another had neurological sequelae (language disorders and epilepsy persisting after a 2-year follow-up).The child who died presented at admission with impaired consciousness, renal failure, pulmonary oedema, and thrombocytopenia (25 x 10^3/mm^3). The child with neurological sequelae presented with hyperparasitemia, impaired consciousness and hypoglycemia.In a univariate analysis, among 422 children, 54 cases were not included due to missing data.versus 0.9, respectively). The average age tended to be higher in children with MTP than in those without MTP .275 children had no need for MTP, while 93 required at least one MTP. The sex ratio (M/F) was comparable regardless of whether or not MTP was used . The average age was comparable in both groups .40 children were admitted to a PICU while 328 children were not. The latter were more likely to be male than the others  was associated with the use of MTPs but not admission to an ICU , specificity (Spe), positive predictive value (PPV) and highest negative predictive value (NPV) are shown in Tables Hyperparasitemia, at the 4% threshold, was not an independent risk factor for the use of MTPs  or PICU admission . We tried to determine the most accurate threshold of parasitemia for predicting severity, defined as the use of MTPs and / or PICU admission. According to the ROC curves , a 4% paVery few data are available on severe pediatric imported malaria. To our knowledge, this study is the largest ever reported.The annual incidence of severe pediatric malaria in France during the period of our study followed the trends observed in Western countries. After a steady increase during the ‘90s, attributed to the increase in air traffic to endemic areas, a gradual decline in incidence was seen after 2000 despite an increasing number of travelers to the areas at risk . This paOf note we observed practitioner’s difficulties in identifying severe malaria, leading to inappropriate therapeutic strategies such as administering antimalarial drugs orally for the treatment of severe cases. This emphasizes the importance of identifying risk factors for severe malaria in pediatric travelers as well as specific prognostic factors in severe cases.The overall lethality was 0.1 per 1000 for pediatric cases reported to the CNR du paludisme between 1996 and 2005. In severe cases reported in targeted hospitals, the case fatality rate was 0.2% (1 death / 422 cases). These results are consistent with the lethality of imported malaria, 0.2% according to data reported from 11 Western countries including France, a rate 10 times lower than in adults with imported malaria .Plasmodium species infections were negatively associated with severe cases.We compared uncomplicated and severe malaria cases to assess risk factors for severe malaria. Several risk factors were positively associated with severe cases: age <2 years, a stay in the Sahel, and thrombocytopenia. Conversely, mixed In endemic countries, it is well established that young children are at high risk for life-threatening malaria .Why travel in the Sahel is a marker of severity is not readily explained. Host or environmental factors might be involved. Further research is needed, including anthropological studies on the perception of malaria risk and adherence to prevention among travelers, of whom most are “visiting friends and relatives” , 5, 17.Thrombocytopenia is a common finding in non-immune children with malaria, whether living in or returning from endemic areas , 11, 18.In our study, a thrombocytopenia <150 x 10^3/mm^3 was significantly more frequent in severe than in uncomplicated cases of malaria. This finding is consistent with pediatric malaria data both in endemic areas , 18–20, Plasmodium species infections were associated with a halving of the risk of severe malaria, compared to monospecific falciparum infections . This has been previously reported both in people living in endemic areas as well as in travelers [P. vivax, a species less life-threatening that P. falciparum, could reduce the severity of falciparum malaria attacks through cross-species immunity. Further studies are needed to confirm this finding, since deaths can occur with mixed P. falciparum—P. vivax infections despite the lack of cytoadherence by P. vivax in brain capillaries [Of note, we found that mixed ravelers , 23. It illaries .Delayed diagnosis  remains a concern in imported pediatric malaria. The average delay reported in previous French studies varied from 4.5 to 9.5 days . In our In endemic areas, delay in seeking care was shown to vary according to the criterion of severity  . This coHyperparasitemia was the most frequent criteria at admission in our study. It was associated especially with neurological disorders or hypoglycemia.The most frequent association of clinical criteria met in our series was neurological disorders and jaundice (N = 7).In this study we assessed the relevance of several criteria of severe malaria updated by the WHO in 2000 in predicting severity. Neurologic disorders, i.e., impaired consciousness, coma and convulsions, were the most reliable prognostic indicators in our series, using either the need for MTPs or PICU admission. Coma is the criterion whose reliability is one of the most consistently reported in the pediatric literature , 27. It Severe anemia is the most common manifestation of severe malaria in children, whatever the transmission level, and is responsible for the highest number of malarial-related deaths in malarial settings , 22, 32.In our series, respiratory distress was strongly predictive for PICU admission (PPV: 85.7%). This major criterion of severity in children should not be confused with the acute respiratory distress syndrome in adults (ARDS). It is of high prognostic relevance in the pediatric literature from tropical countries, whatever the level of malarial transmission , 36.Acidosis is also predictive of death, especially in endemic zones when combined with either cerebral malaria or severe anemia , 38. In falciparum malaria. In the UK, a parasitemia > 2% as a unique criterion of severity is considered to require close monitoring of the child in an ICU [In endemic areas, hyperparasitemia was found to be an independent predictor for cerebral malaria and death, especially at ages below 5 years , 30, 40.n an ICU . In our n an ICU . Children an ICU , 41.Multivariate analysis of severe cases identified other factors which can help practitioners to assess severity: (i) age <2 y: these children had almost 4 times the risk for MTP use than children ≥2 y, but had the same risk for PICU admission compared to older children. This discrepancy may be explained by the higher frequency of some complications occurring below 2 y, i.e., hypovolemia or severe anemia, which can be corrected with procedures not requiring a PICU admission; (ii) a stay in Sahel: this original finding merits further research; (iii) the lack of chemoprophylaxis: this result is consistent with data from adult travelers , 42;  children admitted for severe malaria met the definition of MOD. These children were more frequently admitted to intensive care because they often required several MTPs, e.g., oxygen therapy and transfusions (data not shown).No standardized prognostic score has been validated regarding the need for intensive care in imported pediatric malaria, in contrast to the simplified severity index (SAPS II) in adults . In endeOur study has several limitations: (i) Reporting of imported malaria to CNR du paludisme by the members of this network is voluntary. Thus the cases extracted from the national database for our study may not be entirely representative of the total French experience (ii) Our study is retrospective, with missing data; (iii) The study involved cases from 1996–2005, thus including data reflecting now-outdated practices; (iv) The design of our study, i.e., selected hospitals declaring more than 5 severe pediatric malaria cases throughout the period, missed 55.8% of pediatric severe cases registered by the CNR du paludisme, and; (v) we did not assess all the WHO severity criteria as prognostic factors in our study, given their low frequency in our series and/or missing data.However, we do not think these limitations alter the accuracy of the results. Moreover this study is nationwide over ten years, and is the largest series of imported pediatric malaria to date.Plasmodium species infection are protective against severe malaria. Among severe cases, a stay in Sahel, absence of chemoprophylaxis, age <2 y, presence of the major 2000 WHO criteria of severity, parasitemia 8% or a thrombocytopenia <100 x 10^3/mm^3 were relevant prognostic factors. This study provides important information to help practitioners in the evaluation and management of severe imported pediatric malaria in France. However these findings should be confirmed by prospective studies.Among children with malaria after a stay in endemic zones, those at risk for severe malaria are likely to be infants, had visited a Sahelian country, missed chemoprophylaxis, or to present with thrombocytopenia <100 x 10^3/mm^3. Conversely, mixed


In the advent of a decline in the global incidence of malaria, it is important to generate updated data on the burden of malaria in people living with HIV (PLWHIV). This study was designed to determine the prevalence of malaria in PLWHIV in Yaounde, Cameroon, as well determine the association between CD4+ T cell count was performed using a flow cytometer (BD FASCount™). Giemsa-stained blood films were examined to detect malaria parasite. The Pearson’s chi-square, student’s T-test, ANOVA, and correlation analysis were all performed as part of the statistical analyses.In a cross sectional study performed between April 2015 and June 2016, 355 PLWHIV were enrolled and blood samples were collected for analysis. Complete blood count was performed using an automated haematology analyser  and CDp < 0.001); and in participants who were not on cotrimoxazole prophylaxis (p = 0.002). The prevalence of malaria (p < 0.001) and malaria parasite density (p = 0.005) were observed to be progressively higher in participants with CD4+ T cell count below 200cells/μl. Furthermore, the mean CD4+ T cell count was observed to be lower in participants coinfected with malaria compared to non-coinfected participants (323.5 vs 517.7) (p < 0.001). In this study, a negative correlation was observed between malaria parasite density and CD4+ T cell count (p = 0.019).The prevalence of malaria observed in the study was 7.3 % (95 % CI: 4.8–10.6). No significant association was observed between the prevalence of malaria and age or gender. The prevalence of malaria was higher in participants who were not sleeping in insecticide treated bed nets, ITNs (A low prevalence of malaria was observed in the study population. Some of the factors accounting for the low prevalence of malaria in this study population may include the health seeking habit of PLWHIV, the use of cotrimoxazole based chemoprophylaxis, and their cautious use of ITNs. Plasmodium. There are five species of Plasmodium which cause disease in humans namely; Plasmodium ovale, P. vivax, P. malariae, P. knowlesi and P. falciparum, with the latter being the most virulent accounting for the majority of cases and deaths attributed to malaria. As in most parts of SSA, P. falciparum is the predominant Plasmodium spp. in Cameroon, accounting for almost 100 % of all malaria-related cases [Although the number of cases and death attributed to malaria have witnessed a decline recently, the disease is still responsible for a significant morbidity and mortality especially in sub-Saharan Africa (SSA) and in children below 5 years . In 2015SSA is also the region most affected by the HIV pandemic. In 2014, there were 25.8 million people living with HIV in SSA , account4+ T cells overtime, meanwhile malaria coinfection with HIV results in more episodes of symptomatic malaria [Malaria and HIV are both common in SSA because all the factors which favour transmission are present including poverty. Due to their overlapping distribution, coinfection with malaria and HIV are therefore bound to be common in the area. Coinfection with malaria and HIV is thought to have a synergistic effect, with studies reporting that repeated infection with malaria leads to a more rapid decline in CD malaria , and mor malaria –12. The  malaria , 14.4+ T cell count and malaria. This will generate updated baseline data for clinico-epidemiologic purposes which will improve upon the management and control of both diseases.Although the prevalence of coinfection between malaria and HIV had earlier been reported in some parts of Cameroon , 15, theAnopheles gambiae as the main vector [This study was performed in Yaounde. Yaounde (3°52′N 11°31′E) with an average elevation of 750 m, is the capital of Cameroon. With a population of roughly 2.5million, Yaounde is second only to Douala as the largest city in Cameroon . Yaoundén vector . Accordin vector .This was a cross-sectional study performed between April 2015 and June 2016, involving PLWHIV. Participants were recruited at the Yaounde Military Hospital (YMH). The YMH is one of the main treatment facility for HIV/AIDS patients in Yaounde receiving close to a thousand HIV/AIDS patients every month, coming from all over Yaounde and it’s environ.Using the following formula for sample size calculation ;\documenZ = 1.96p = prevalence of malaria among PLWHIV = 29.4 % .e = error rate = 0.05It was estimated that we will require a sample of at least 319 PLHIV.A convenient sampling technique was used, where PLHIV were consecutively recruited into the study as they came for consultation or to collect their ARV drugs.PLWHIV were recruited at the HIV treatment center of the Yaounde Military Hospital. The study population consisted of HIV/AIDS patients on treatment and newly diagnosed cases (treatment-naïve). To be included in the study, participants were to be male or female of all ages. Participants were not to be on any antimalarial medication one week prior to the study. Participants were also required to provide a written informed consent, which was duly explained to them in English, French or the local Pidgin English. For minors and those who could not sign the informed consent, their parents, guardians, or next of kin did on their behalf.4+ T cell count. Thick and thin blood films were also prepared for malaria microscopy.Once participants gave their informed consent, their blood samples were collected. About 4 ml of blood was collected into EDTA anticoagulated test tubes following aseptic techniques, to perform the complete blood count (CBC) and CDThe CBC was performed using the Mindray® automatic haematology analyser .4+ T cell counts were determined using a flow cytometer, BD FASCount™.CDThick and thin blood films were prepared and stained with 10 % Giemsa and examined using methods previously described . Blood fT-test and ANOVA for the comparison of group means, and correlation analysis to determine the association between parasite density and CD4+ T cell count. Statistical significance was set at p < 0.05.Data collected were entered into an Excel spreadsheet and analysed using the Stata® version 12.1 software . The statistical tests performed included the Pearson’s Chi-square for comparison of proportions, the Student’s Four hundred and seven PLWHIV were approached, 355 of them meet the inclusion criteria and were therefore enrolled PLWHIV successfully took part into the study. Among them were 236 (66.5 %) females and 119 (33.5 %) males (Table p = 0.960). The prevalence of malaria was higher in males compared to females (Table p = 0.388). The prevalence of malaria was significantly higher in participants that were not sleeping under ITNs compared to those who were (p <0.001). The prevalence of malaria was also significantly higher in participants who were not on prophylaxis with cotrimoxazole (p = 0.002) (Table Twenty six 26) of the 355 PLWHIV were infected with malaria parasite giving a prevalence of 7.3 % (95 % CI: 4.8–10.6). The prevalence of malaria was higher in individuals between 40 and 59years of age (Table 6 of the 4+ T cell count of the participants ranged between 38 and 1600 cells/μl (mean ± SD = 503.5 ± 276.2). The prevalence of malaria parasitaemia was higher in participants with CD4+ T cell count below 200 cells/μl and lowest in participants with counts above 500cells/μl (Table 4+ T cell count (p < 0.001). Furthermore, the mean CD4+ T cell count was lower in patients coinfected with malaria parasites compared to non-coinfected participants (323.5 vs 517.7). The difference in the CD4+ T cell counts between malaria coinfected patients and non-coinfected patients was observed to be significant statistically (p < 0.001).The CD4+ T cell count below 200 cells/μl . Furthermore a negative correlation was observed between CD4+ T cell count and GMPD   observed was 1219.7 parasites/μl. The GMPD was progressively higher in CD4+ T cell count and malaria.Due to the overlapping distribution of malaria and HIV, coinfection between malaria and HIV are therefore common in SSA. With the declining trend in the global incidence of malaria , it is ip = 0.002). Trimethoprim-Sulfamethoxazole which is the active ingredient of CTX is also effective therapeutically against malaria [In the current study. A prevalence of 7.3 % for malaria parasite was observed. This prevalence is lower compared to the prevalence of 34 % for malaria reported in the general population of Yaounde . This pr malaria , 27. A s malaria . Regular malaria  in whichp = 0.960) or gender (p = 0.388). These findings echo earlier reports by Njunda et al. [p = 0.540). This finding is in resonance with the study by Kimbi et al. [In the current study, no significant association was observed between the prevalence of malaria and age . This is contrary to the study performed by Iroezondu et al. [4+ T cell count was significantly lower in HIV patients coinfected with malaria compared to those without, which confirms the study by Tay et al. [4+ T cell count. The finding of a significant association between malaria prevalence and CD4+ T cell count is contrary to studies performed elsewhere [Overall, the mean CDu et al. . Furthery et al.  in whichlsewhere , 25.4+ T cell count below 200cells/μl (p = 0.005). This increasing parasite density with decreasing CD4+ T cell count could be attributed to the weakening of the immune responses of these patients. Both the cellular and humoral immune responses are thought to play a role in the protection against malaria [4+ T cell. A depletion in the number of CD4+ T cells will therefore affect the cellular and humoral immune responses and consequently the immune responsiveness to malaria antigens [The GMPD observed in this study was 1219 parasites/μl. The GMPD was also observed to be progressively higher in patients with CD malaria , and theantigens , 32. As In the current study, only participants who attended the HIV/AIDS treatment center of the Yaounde Military Hospital were recruited. This may be biased against PLWHIV do not attend the facility or who did not come to the facility within the study timeframe. This may not give a true representation of PLWHIV in Yaounde. This serves as a major limitation of the study. In addition, the study was performed during the rainy season, a period well known for high malaria transmission. This also, may have affected the true representation of the prevalence of malaria in the study population, as data collected during the dry season may be lower. Studies to determine how the prevalence of malaria among PLWHIV varies with respect to season will therefore be needed in the study area.4+ T cell count below 200cells/μl, as well as a negative correlation between the parasite density and the CD4+ T cell count is indicative of the role of the immune response in the protection against malaria in the target population.A low prevalence of malaria was observed among PLWHIV in Yaounde, which is lower compared to prevalence reported in the general population of Yaounde. The prevalence was also lower compared to the prevalence reported in other countries in Sub-Saharan Africa. The low prevalence of malaria could be attributed to the wide use of insecticide treated bed nets and the cotrimoxazole based chemoprophylaxis in the study population. The prevalence of malaria parasitaemia was not observed to be associated with age or gender. The findings of a high prevalence of malaria and a higher parasite density in participants with CD


Plasmodium, P. vivax is commonly found in temperate-zone countries including South Korea. In this article, we first review the history of malarial infection in Korea by means of studies on Joseon documents and the related scientific data on the evolutionary history of P. vivax in Asia. According to the historical records, malarial infection was not unusual in pre-20th-century Korean society. We also found that certain behaviors of the Joseon people might have affected the host-vector-pathogen relationship, which could explain why malarial infection prevalence was so high in Korea at that time. In our review of genetic studies on P. vivax, we identified substantial geographic differentiation among continents and even between neighboring countries. Based on these, we were able to formulate a strategy for future analysis of ancient Plasmodium strains in Korea.Malaria, one of the deadliest diseases in human history, still infects many people worldwide. Among the species of the genus Anopheles mosquito. The Plasmodium parasite has a complex life-cycle of sexual reproduction inside the mosquito vector and asexual stage in the vertebrate host. In brief, malarial sporozoites are inoculated into human hosts when a mosquito bites them [Globally, malaria is the fifth deadliest disease, infecting approximately 200 million people worldwide –3. Malartes them . After ates them . Some oftes them . Sexual tes them . Plasmodium are known to cause malaria: P. falciparum, P. vivax, P. malariae, P. ovale, and P. knowlesi [ P. simium from monkey [ P. vivax and P. falciparum are the most commonly detected causative pathogens of human malaria. Clinical manifestations of uncomplicated malaria are nonspecific: headache, fever, malaise, myalgia, nausea, vomiting, and abdominal pain. Rare cases of malaria show severe manifestations including anemia, thrombocytopenia, pulmonary edema, renal failure, hepatic dysfunction, and splenic rupture [In general, five species of genusknowlesi . Recent m monkey . Among t rupture . P. falciparum's symptoms are serious enough to show the highest mortality rate whereas P. vivax, P. malariae, and P. ovale exhibit generally nonfatal clinical courses [ Plasmodia subtype differs as well. P. falciparum is more prevalent in tropical or subtropical zones including Sub-Saharan areas but relatively absent in temperate countries [ P. vivax generally infects human populations in temperate and tropical zones but is not so prevalent in Sub-Saharan Africa [ P. vivax malaria was endemic even in some high latitude countries  at certain points in history [Although malarial species share typical signs and symptoms such as undulating intermittent fever, they also have different traits depending on each subtype. courses . The geoountries . Meanwhin Africa , 3, 9. P history , 10, 11. vivax malaria was generally known to induce anemia, further causing porotic hyperostosis (PO) or cribra orbitalia (CO) in the cranium [ Plasmodium infection is not the only cause of them [As malaria historically has been, and continues to be, one of the most serious diseases, it has attracted the attention of many paleopathologists. Studies on ancient malarial infection have been conducted by various methods such as osteoarchaeological and biomedical approaches . As chro cranium , anthrop cranium , 12–15.  of them . Other p Plasmodium aDNAs reportedly have been obtained from Egyptian mummies [In recent years, the paleopathological study of malaria has been revolutionized by successful applications of immunological and ancient DNA (aDNA) analyses to archaeological specimens. To detect malaria-related proteins, researchers performed the dipstick assay or new-generation immunoassays on ancient mummies–19 or sk mummies , 22–24,  mummies , 15th-to mummies , and 1st mummies . As a pa mummies . Plasmodium infection. Nonetheless, most of the requisite paleopathology still remains to be revealed in Asia as few medical studies on the ancient malaria have been reported in the area. Herein, then, we offer this historical review as a fundamental basis for future research of ancient malaria infection in Korea and other Asian countries.Although immunological and molecular analyses have become more reliable tools for the study of ancient malaria, the data obtained to date are not sufficient in terms of quantity and quality . Moreove P. falciparum was transmitted from chimpanzees and gorillas in Africa [ P. vivax originated from another nonhuman primate, possibly macaques, in Southeast Asia [ P. falciparum and P. vivax originated in Africa and that P. vivax transmission to human beings might have occurred much earlier than P. falciparum did by a host switch [Parasitologists have speculated that human malaria might have been transmitted from nonhuman primates by a host-switch event . Initialn Africa  while P.ast Asia , 32, 33.t switch . P. vivax populations existed between 50 and 550 ka before the present [ P. vivax genome analysis, the global population size of vivax malaria might have expanded slowly until about 60 ka BP, which is closely consistent with the demographic history of mankind [ P. vivax populations occurred at about 51 ka BP, the latter appears to have undergone a rapid exponential increase in population size [According to the estimated time to most recent common ancestor (TMRCA), the ancestor of the extant present . In a de mankind , 35. Oncion size , 37. It  P. vivax worldwide, two divergent groups were identified: a large star-like cluster and a divergent cluster [ “Asia a” of Central China and “Asia b” of China, Korea, and Indonesia. The divergent East Asian P. vivax lineage was connected to the large star-like cluster by a group of haplotypes found in Southeast Asia [ P. vivax might have been split from all other vivax malaria and developed a distinct demographic history for at least 121 ka [ P. vivax-resistant RBCs (Duffy-negative phenotype) occurred in Sub-Saharan peoples [ P. vivax to East Africa by sea-going traders from Asia [In the phylogenetic tree of cluster . The latast Asia . Based ot 121 ka . Meanwhi peoples . Due to rom Asia .The historical record is important for understanding the pattern of malarial infection in ancient civilizations. In the  P. vivax [ Plasmodium subtype. Notwithstanding the benign traits of P. vivax, relapsed infection typically might have exhausted people, often eventually killing them, as seen in similar clinical reports today [Malaria must have been endemic in East Asia from ancient times as well, as descriptions about malaria-like symptoms can be seen in Chinese historical records . AlthougP. vivax , the Josts today , 49–53. First Annual Report of the Korean Government Hospital, Seoul, Dr. Horace Newton Allen described “endemic intermittent fever”  as the most commonly observed sign among Korean patients who visited his hospital [Before the first modern medical record on malaria in Korea (1886), prevalences of malarial infection could not be reliably calculated. In thehospital , 55. Acchospital , 56–63.In malariology, however, the waxing and waning of malarial infection in a specific area cannot be explained so simply. In addition to wetlands, environmental alteration or degradation due to human activity also has a great influence on the density and activity of mosquito populations and, further, on the prevalence of malaria itself–66.  TabWe do not yet know whether the close relationship between environmental change by agriculture and malarial infection is a universal phenomenon beyond certain temporal and spatial limits. In a recent cross-national analysis, however, correlations among anthropogenic activity, mosquito population sizes, and malaria rates were seen to have been common in many parts of the world . The finIn brief, the 15th to 19th centuries in Korean history were a turbulent and dynamic period during which the Joseon people were highly motivated to be involved in agricultural innovation, thereby eventually effecting major changes in their sociocultural environment . By infuOn such lands, Joseon farmers planted crops. Rice was very popular, becoming the most preferred crop by the late Joseon Dynasty . To meet Plasmodium infection [ Plasmodium infection in Asia [From the late 19th century, the diagnosis and treatment of malaria began to be performed by specialists in Western medicine. In 1913, for example, an intermittent fever observed among Korean patients was finally confirmed by a modern microbiology technique to benfection , 81. Dur in Asia . Whereas in Asia .However, we must also consider the possibility that malarial prevalence in the country was not reduced by antimalarial medications or insecticides alone. This idea is supported by instances in European history. In the 18th to 19th centuries, malaria was still prevalent in Europe, though it rapidly declined thereafter, finally disappearing from most regions by the 1930s . The ret Anopheles mosquito migrating from the north [Unfortunately, the reemergence of malaria after long-term eradication is not a rare phenomenon in the world, 88. In he north , 90. In he north . Today, he north , 90, 91.Malarial infection in history often cannot be fully evidenced by the examination of historical documents. This is due to difficulty in accurate diagnosis of ancient malaria cases in history. Actually, the signs and symptoms of ancient malaria patients were often vaguely described in historical literatures by modern clinical medicine standards. In addition, diagnosis of ancient malaria (solely) by the examination of archaeologically obtained skeletal remains is also highly problematic as malaria leaves little traces on bones. In this regard, we note that DNA-based study could be useful for acquiring scientific evidences of specific diseases prevalent in history. P. vivax is generally targeted on the protozoan parasites' surface proteins by which the erythrocyte invasion of the vivax-malarial parasite can be triggered [ P. vivax merozoite surface protein (PvMSP), which is abundantly expressed on the merozoites of vivax malaria [ P. vivax merozoites and that plays a crucial role in RBC invasion of parasites [ P. vivax into human RBC[DNA analysis ofriggered , 93. One malaria . Duffy-barasites , 96. As human RBC, PvMSP ahuman RBC, 97–99 thuman RBC–103. P. vivax in South Korea [ P. vivax isolates, as the outcomes would doubtlessly be useful for understanding the nature of the P. vivax population in South Korea [ P. vivax is higher than that of P. falciparum, suggesting that the former has a long, complex, but successful evolutionary history of adaptation [ P. vivax reemerged in South Korea, the isolates of the years 1993 to 2000 were genetically closely related, meaning that its genetic diversity was very low at the initial stage of its reintroduction [Since the reemergence ofth Korea , 87, Korth Korea , 103–108th Korea , 91, 97.aptation , 109–112oduction . Since 2oduction , 97, 108oduction . P. vivax isolates. Among them, SK-3 was a new clade that had not been identified at the early phase of reemergence in the same Korean isolates [ P. vivax isolates has been under the strong influence of positive natural selection [Ju et al.  also repisolates  but lateisolates . They agelection   of spongy bones inside vertebrae (possibly containing hemopoietic cell remains) was chosen commonly as specimens for aDNA analysis of malaria; and in another case, first or second molars have been selected foranalysis . Many fuWith respect to human samples obtained from archaeological sites, scientific techniques can be done to reveal whether the individual had suffered from malaria in his lifetime or to obtain phylogenetic information of its ancient genome. As the previous studies on ancient malaria have focused mainly on specimens from Egypt and Europe, however, the current information so far obtained carries a serious geographical bias. More extensive geographic samplings and assays are thus needed in order to obtain a more comprehensive demographic evolutionary history of malaria. P. vivax there and elsewhere in Asia. To those ends, we first examined the historical-documentary evidence of ancient malarial outbreaks in Joseon society and found that malarial epidemics were in fact not unusual in pre-20th-century Korea. We detected changes in the host-vector-pathogen relationship, which probably affected the proliferation of the mosquito vector and indeed the prevalence of ancient malaria in Joseon society. We also noted, in our review of genomic studies on P. vivax, substantial geographic differentiation of vivax-malarial DNA between different continents and even neighboring countries. Many scientific studies on the history of malaria will be done with ancient specimens in Korea and Asia, pending the permission of the relevant medical-ethics review boards.Like the other continents, Asia is a region wherein malarial infection has been epidemic in history. Nonetheless, very little has been done in the way of relevant paleopathological studies on ancient malaria. We thus reviewed the history of malaria in Korea and attempted to derive scientific clues to the evolution of


TOLLIP polymorphisms (rs5743899 and rs3750920) were reported to be influencing IL-6, TNF and IL-10 expression. In this study, TOLLIP variants were investigated to their relation to Plasmodium vivax malaria in the Brazilian Amazon.Toll-interacting protein is a negative regulator in the TLR signaling cascade, particularly by impeding the TLR2 and, TLR4 pathway. Recently, TOLLIP was shown to regulate human TLR signaling pathways. Two common P. vivax malaria and, 263 healthy controls with no previous history of malaria were included in the study. Genomic DNA was extracted from blood collected on filter paper, using the QIAamp® DNA Mini Kit, according to the manufacturer’s suggested protocol. The rs5743899 and rs3750920 polymorphisms of the TOLLIP gene were typed by PCR–RFLP.This cohort study was performed in the municipalities of Careiro and, Manaus, in Western Brazilian Amazon. A total of 319 patients with 2 = 0.037; D′ = 0.27).Homozygous individuals for the rs3750920 T allele gene had twice the risk of developing malaria when compared to individuals homozygous for the C allele . In the dominant model, carriers the C allele indicates protection to malaria, carriers of the C allele were compared to individuals with the T allele, and the difference is highly significant . The linkage disequilibrium between the two polymorphisms was weak (rTOLLIP rs3750920 T allele with susceptibility to malaria further provides evidence that genetic variations in immune response genes may predispose individuals to malaria.These findings suggest that genes involved in the TLRs-pathway may be involved in malaria susceptibility. The association of the  Anopheles darlingi. In the State of Amazonas, 75,575 cases were reported in 2015. Plasmodium vivax accounts for 90% and, Plasmodium falciparum is responsible for the majority of the remaining cases  was applied for comparison of patients with malaria to healthy controls. Allelic and genotypic frequencies were calculated by gene counting directly. Hardy–Weinberg equilibrium (HWE) was calculated by χ2 test by comparing the observed number of different genotypes frequency with those expected under HW expectation. The software Haploview 4.2 was used for calculating Linkage disequilibrium between the rs5743899 and, rs3750920.Statistical analysis was performed using the website P. vivax and/or P. falciparum, indicating that most individuals were exposed to the malaria parasite along the years. The baseline characteristics of the individuals participating in this study are shown in Table Previous study of surveillance in the Careiro, Brasileirinho, Ipiranga and, Puraquequara communities showed that a significant proportion of the population reported previous infection with TOLLIP gene. The distribution of genotypes and, allele frequencies of both polymorphisms are shown in Table 2 and, D′ are 0.037 and, 0.27 respectively.These individuals were genotyped for the rs5743899 and, rs3750920 of P. vivax-infection and ranges from 1 to 158,777 copies/µL. Low parasitaemia was considered to be equal or below to the median (183 copies/µL). None of the genotypes correlated to either low or high parasitaemia as shown in Table The study examined whether the genotypes of both SNPs were correlated with parasitaemia. Parasitaemia was determined as elsewhere by quantitative Real-Time PCR . ParasitPlasmodium-infected individuals progress to clinical manifestations while the rest remains asymptomatic. Many may also have recurrent infections or malarial episodes in contrast to their neighbors. Altogether, these suggest that the host genetics may play an important role in the clinical outcome and, the identification of genes involved in either susceptibility or resistance to Plasmodium-infection is of utmost interest to the molecular understanding of the disease. Vaccine design may take into account the host genetics.Despite an increasing amount of research on malaria, the molecular mechanism that influences the clinical outcome of the disease is poorly understood. In endemic areas, only a fraction of Plasmodium GPI is recognized by various cell surface TLRs [Plasmodium infection by the host requires a well tune immune response sufficient to restrict the multiplication of the parasite and also to avoid an excess activation of the TLRs intracellular signaling that may lead to an exacerbation of pro-inflammatory cytokines such as TNF, IL-6 and, IFN-γ injuring the host tissue.TLRs upon recognizing invading pathogens orchestrate innate immune responses through the induction of chemokines and inflammatory cytokines to check the parasite. Several polymorphisms present in TLRs genes and, genes involved in its pathways are associated with various infectious diseases , 27. Plaace TLRs  and, proace TLRs . A perfeTOLLIP gene that is a negative regulator of TLRs signaling, are associated with tuberculosis in a Vietnamese population [Two polymorphisms, rs5743899 and rs3750920, in the pulation . The curpulation  and, witpulation , 28 but TOLLIP diverts the pro-inflammatory cytokine response after TLR-2 and, TLR-4 signaling into an anti-inflammatory response that is characterized by an increased IL-10 and, a decreased IL-6 and TNF expression in peripheral blood monocytes . One stuThis study has several limitations. Firstly, the sample size of the study participants is small and although the level of the association with malaria is high, it needs validation with a larger sample size. Furthermore, it can be argued that the association to malaria may be spurious as this Amazonian population is a miscegenation of African, European and, Amerindian origin. The controls were properly selected to match the patients’ ethnicity and, are from the same area of endemicity as the patients. Moreover, the frequency of the genotypes of rs5743899 which is very close to rs3750920 was similar in both patients and controls ruling out a spurious association. Secondly, there were no patients with different clinical manifestations, such as severe, mild and asymptomatic malaria, to perform intra-comparison to denote which allele is related to severe malaria. Lastly, plasma cytokines levels were not assayed to correlate with the genotypes of the SNPs.TOLLIP rs3750920 T allele with susceptibility to malaria further provides evidence that genetic variations in immune response genes may predispose individuals to malaria. Further studies are needed in other vivax regions to confirm whether this allele can be used as a prospective genetic marker for vivax malaria in endemic areas in the future.The present findings suggest that genes involved in the TLRs-pathway may be involved in the pathogenesis of malaria. TOLLIP interacts with TLR-2 and, TLR-4 to skew the pro-inflammatory to anti-inflammatory response. The association of the


P. falciparum merozoites that is essential for invasion of erythrocytes and are vaccine candidates. We determined IgG subclass responses to these proteins among malaria-exposed individuals in Papua New Guinea and their association with protection from malaria in a longitudinal cohort of children. Cytophilic subclasses, IgG1 and IgG3, were predominant with limited IgG2 and IgG4, and IgG subclass-specific responses were higher in older children and those with active infection. High IgG3 to PfRH5 and PfRipr were significantly and strongly associated with reduced risk of malaria after adjusting for potential confounding factors, whereas associations for IgG1 responses were generally weaker and not statistically significant. Results further indicated that malaria exposure leads to the co-acquisition of IgG1 and IgG3 to PfRH5 and PfRipr, as well as to other PfRH invasion ligands, PfRH2 and PfRH4. These findings suggest that IgG3 responses to PfRH5 and PfRipr may play a significant role in mediating naturally-acquired immunity and support their potential as vaccine candidates and their use as antibody biomarkers of immunity.Understanding the targets and mechanisms of human immunity to malaria is important for advancing the development of highly efficacious vaccines and serological tools for malaria surveillance. The PfRH5 and PfRipr proteins form a complex on the surface of  Plasmodium falciparum malaria is a leading cause of morbidity and mortality globally, particularly in young children2P. falciparum vaccine that is capable of generating protective immunity against malaria345673P. falciparum blood stage antigens have been shown to be an important part of this protective immune response3481011121314P. falciparum reticulocyte binding-like homologue protein (PfRH5)16P. falciparum merozoites and includes PfRH1, PfRH2a, PfRH2b, PfRH4 and PfRH5. While all five members of this family play important roles in merozoite invasion, only PfRH5 appears essential to blood stage replication of P. falciparum in vitro131920P. falciparum PfRH5 interacting protein)231322A promising emerging vaccine candidate is P. falciparum in malaria endemic regions of Africa and Papua New Guinea have been shown to develop anti-PfRH5 antibodies526P. falciparum growth in vitro26P. falciparum infection on acquisition of these antibodies and their relationship to the acquisition of antibodies to other PfRH invasion ligands.Individuals naturally exposed to The study population and details of the cohort study, conducted between June and December 2004, have been described previouslyEthics approval was obtained from the Medical Research Advisory Committee, PNG, the Walter and Eliza Hall Institute of Medical Research Ethics Committee, and The Alfred Human Research Ethics Committee and the study was conducted in accordance with the protocol and relevant guidelines. Written informed consent was obtained from all study participants or their guardians (for children).Recombinant PfRH5 and PfRipr were expressed in a wheat germ cell-free expression system (WGCF)ELISA (Enzyme Linked Immunosorbent Assays) were performed in 96-well plates  using established methods described elsewhere10P. falciparum malaria was defined as a fever (>37 °C) and a parasitemia >5000/μlP. falciparum malaria, with subjects stratified as high, medium or low responders, and a log rank test was performed to compare response tertiles. Cox proportional hazard models were used to determine the association between antibody response categories and risk of symptomatic P. falciparum malaria adjusting for potential confounders6a priori), such as parasitemia at enrolment, and red blood cell genetic polymorphisms, were not significantly associated with the outcome of malaria. Age was used as binary variable , as previous studies indicated that this stratification was the most informative approach for assessing the effect of age6Kruskal-Wallis and chi-squared tests were used to determine the association of continuous and categorical variables, respectively, with categorical variables. Correlations between antibody responses were assessed using Spearman’s rho (ρ). To assess the association between subclass responses and protection from symptomatic malaria, children were classified into three equal groups of high, medium and low responders, according to the OD of antibody responses; children who were negative for antibodies were included in the low responder group fever >3 °C and aTo determine the predominant IgG subclass response to PfRH5 and PfRipr, a selection of malaria exposed PNG adults (n = 24) who were resident in Madang Province were tested. IgG1 and IgG3 were the predominant subclass response to both antigens . IgG3 was the predominant subclass response against both antigens with a higher seroprevalence compared to IgG1 (p < 0.001) .In the children’s cohort, IgG1 and IgG3 to PfRH5 and PfRipr had higher median ODs compared to malaria-naïve negative controls indicating the specificity of <9 yrs: 38.5%, p < 0.001; PfRipr >9 yrs: 50.0%, <9 yrs: 38.5%, p = 0.07). Higher seroprevalence of IgG1 and IgG3 subclass responses were also found in older compared to younger children for both PfRH5 and PfRipr  (To investigate the relationships between age and IgG subclasses responses (IgG1 and IgG3) to PfRH5 and PfRipr, seroprevalence was compared between younger and older children . The ser = 0.01) .P. falciparum infection generally have higher levels of antibodies to merozoite antigens46Previous studies have shown individuals with active Responses to PfRH5 were significantly positively correlated with each other . Total IP. falciparum infection.To investigate the co-acquisition of antibodies to different antigens, the total IgG and subclass responses to PfRH5 and PfRipr were compared to previously published data on IgG responses to other PfRH family proteins; PfRH2 , there were significant differences between the three IgG subclass groups, with high responders having a longer time to first symptomatic P. falciparum episode than medium and low responders for both PfRH5 and PfRipr  (To investigate the association between antibody responses and protection from symptomatic < 0.001) .P. falciparum malaria compared to individuals with low IgG levels, as reported previouslyP. falciparum malaria after adjusting for potential confounders, as previously reportedP. falciparum malaria after adjusting for confounders . Associations were weaker for IgG1 to PfRipr, and did not reach statistical significance after adjusting for confounders .After adjusting for the confounding effects of age and location of residence, individuals with high levels of total IgG to PfRH5 had a 65% lower risk of symptomatic In this study we found that cytophilic antibodies IgG1 and IgG3 were the predominant subclass responses to PfRH5 and PfRipr, which are present as an invasion complex on the merozoite surface. Interestingly, IgG3 responses were most strongly associated with protection from malaria in a longitudinal cohort study of children, whereas protective associations for IgG1-specific responses were weaker and did not reach statistical significance after adjusting for confounding variables. These findings provide further support for PfRH5 and PfRipr as malaria vaccine candidates and suggest that the IgG3 response is more important in mediating protection.P. falciparum among individuals, and host factors4647et. alP. falciparum infection compared to uninfected children. The overall trend of increased IgG subclass responses in those with concurrent parasitemia is consistent with other studies and reflect potential boosting of antibody responses with infection634Our initial studies in a selection of adults indicated that IgG1 and IgG3 were the predominant responses to PfRH5 and PfRipr, with little IgG2 and IgG4 reactivity. In the larger children’s cohort, both IgG1 and IgG3 were prevalent, but IgG3 responses were significantly more common for both antigens. A prior study in Senegal found that IgG1 responses were more prevalent than IgG3 among adults (69% vs. 31% respectively), although the sample size in that study was small (n = 19)P. falciparum malaria during follow-up. There was evidence of a dose-response effect, as the risk of symptomatic P. falciparum malaria was significantly reduced in those with high compared to medium and low antibody levels. Protective associations were substantially stronger for IgG3 responses to PfRH5 and PfRipr than IgG1 responses, and only the associations between IgG3 and protection remained significant after adjustment for potential confounders. Interestingly, previous studies of IgG subclass responses to other members of the PfRH invasion ligand family found that IgG3 responses were strongly associated with protection from malaria746A strength of this study is the longitudinal study design that enabled prospective examination of the association between antibodies and subsequent malaria episodes. We found that higher levels of IgG1 and IgG3 against PfRH5 and PfRipr at enrolment were associated with protection from subsequent symptomatic Defining the nature of IgG subclass responses to PfRH5 and PfRipr, and their associations with protective immunity, is important because different subclasses have different immune effector functions371537P. falciparum infection, supporting a view that immunity to malaria is mediated by a repertoire of antibodies to multiple invasion ligands.Total IgG, IgG1 and IgG3 to all PfRH family proteins were correlated, with the highest correlations observed with the predominant IgG subclass responses. IgG1 and IgG3 responses to PfRH5, or PfRipr, were found to be significantly correlated with each other. Furthermore, IgG1 and IgG3 to other PfRH family proteins, PfRH2 and PfRH4, were also correlated with PfRH5 and PfRipr responses. The predominant IgG subclass responses for each antigen were most strongly correlated. These findings indicate the co-acquisition of antibodies to multiple PfRH invasion ligands following exposure to P. falciparum malaria, finding that IgG3-specific responses are most strongly associated with immunity. This knowledge provides important insights into potential mechanisms mediating protection, and identifies possible antibody biomarkers of protective immunity that may have important applications for sero-surveillance in P. falciparum malaria endemic areasThis is the first study to examine the association of naturally-acquired IgG subclass responses to PfRH5 and PfRipr with protection from symptomatic How to cite this article: Weaver, R. et al. The association between naturally acquired IgG subclass specific antibodies to the PfRH5 invasion complex and protection from Plasmodium falciparum malaria. Sci. Rep.6, 33094; doi: 10.1038/srep33094 (2016).


Plasmodium vivax infections in humans or in new world monkeys pose research challenges that necessitate the use of alternative model systems. Plasmodium cynomolgi is a closely related species that shares genetic and biological characteristics with P. vivax, including relapses. Here, the haematological dynamics and clinical presentation of sporozoite-initiated P. cynomolgi infections in Macaca mulatta (rhesus macaques) are evaluated over a 100-day period.M. mulatta were inoculated with 2000 P. cynomolgi B strain sporozoites. Parasitological and haematological data were collected daily to study the clinical presentations of primary infections and relapses. Peripheral blood and bone marrow aspirates were collected at specific time points during infection for future and retrospective systems biology analyses.Five Patent infections were observed between days 10 and 12, and the acute, primary infection consisted of parasitaemias ranging from 269,962 to 1,214,842 parasites/µl (4.42–19.5 % parasitaemia). All animals presented with anaemia, ranging from moderate (7–10 g/dl) to severe (<7 g/dl), based on peripheral haemoglobin concentrations. Minimum haemoglobin levels coincided with the clearance of parasites and peripheral reticulocytosis was evident at this time. Mild thrombocytopaenia  was observed in all animals, but unlike haemoglobin, platelets were lowest whenever peripheral parasitaemia peaked. The animals’ conditions were classified as non-severe, severe or lethal (in one case) based upon their clinical presentation. The lethal phenotype presented uniquely with an exceptionally high parasitaemia (19.5 %) and lack of a modest reticulocyte release, which was observed in the other animals prior to acute manifestations. One or two relapses were observed in the four surviving animals, and these were characterized by significantly lower parasitaemias and minimal changes in clinical parameters compared to pre-infection values.P. cynomolgi B strain sporozoites recapitulated pathology of human malaria, including anaemia and thrombocytopaenia, with inter-individual differences in disease severity. Importantly, this study provides an in-depth assessment of clinical and parasitological data, and shows that unlike the primary infections, the relapses did not cause clinical malaria. Notably, this body of research has provided experimental plans, large accessible datasets, and blood and bone marrow samples pertinent for ongoing and iterative systems biology investigations.Rhesus macaque infections initiated by The online version of this article (doi:10.1186/s12936-016-1480-6) contains supplementary material, which is available to authorized users. Plasmodium falciparum is responsible for the largest number of cases and malaria-associated deaths, with extensive research efforts focused on understanding the basic biology and associated pathogenesis of this parasite species. Plasmodium vivax has also been recognized as a major contributor to the global burden of malaria, and numerous recent reports indicate infections can result in complications with lethal outcomes [P. vivax is apparent, greatly due to the challenges associated with studying this species, which include the lack of a long-term in vitro culture system or a rodent model that can support the entire life-cycle and re-create critical aspects of P. vivax infections and disease as observed in humans [Malaria represents a constant burden and public health challenge in approximately 100 countries worldwide [outcomes –5. Howevn humans –9.Plasmodium vivax, as well as Plasmodium ovale, differ from the three other human malaria-causing species  because they can develop dormant liver-stage forms, known as hypnozoites [nozoites . Hypnozonozoites . It is cUnderstanding the course of primary and relapse infections, and specific mechanisms that result in vivax malaria pathogenesis, disease severity and recovery, are important goals. In particular, the identification of mechanisms that function in the peripheral blood and bone marrow resulting in the onset and recovery of malarial anaemia , 13 and Plasmodium vivax can be studied directly in New World monkey species, such as squirrel (Saimiri boliviensis) and owl (Aotus sp.) monkeys, and these models have been important, for example, in testing vaccine and drug candidates with parasite challenge infections [P. vivax infected erythrocytes [Non-human primate models have contributed to the broad understanding of malaria biology, pathogenesis and immunity with regards to liver-stage and blood-stage infections, and they have also been instrumental for screening vaccine and drug candidates or specific formulations –21. Plasfections , 23 and,hrocytes , 25. HowPlasmodium cynomolgi is a simian malaria parasite of Old World macaques that is genetically closely related to P. vivax [Plasmodium infection studies that require repeated sampling within a short time frame. Macaca mulatta (rhesus monkey) and M. fascicularis genome sequences [P. cynomolgi [P. vivax , 27 and P. vivax , 29, devP. vivax , 31, andP. vivax , 32, 33.P. vivax . Young aequences , 36 and ynomolgi , have beP. cynomolgi in rhesus macaques are presented with the long-term goal of using this model for systems biology investigations to better understand human malaria pathogenesis, particularly as it pertains to pathophysiological complications observed in sick patients and with regard to the impact of relapses on both the health of individuals and transmission [P. cynomolgi-macaque model to understand the parasite’s basic biology and parasite kinetics, including the study of hypnozoites and relapses [Here, comprehensive analyses of sporozoite-initiated, longitudinal infections of smission . Previourelapses , 33, 38.The basic experimental design of this study is shown in Fig. In addition to collecting samples for analysis at major points during the infections, this study was also designed to collect clinical data on a daily basis. The aim of these collections was to develop highly resolved clinical kinetics. To achieve this goal, the animals were accessed daily without sedation for obtaining up to 100 µl of blood through standardized ear-prick procedures, similar to the collection of blood from a human finger-prick. This blood was used to perform complete blood counts (CBCs) as well as enumerate reticulocytes and parasites during the infection.M. mulatta  born and raised at the Yerkes National Primate Research Center (YNPRC), an Association for Assessment and Accreditation of Laboratory Animal Care (AAALAC) international-certified institution, were assigned to this study. The animals were socially housed in pairs at YNPRC during the experiment, and all housing was in compliance with Animal Welfare Act regulations as well as the Guide for the Care and Use of Laboratory Animals. Males were designated to eliminate confounding factors studying anaemia that could be attributed to loss of blood during the female menstrual cycle. An additional rhesus monkey was assigned at the Centers for Disease Control and Prevention (CDC) and utilized for the generation of sporozoites. Standard procedures for monitoring the clinical conditions of the animals, collecting biological samples (venous blood and bone marrow aspirates), and performing mosquito feedings on infected animals to generate infectious salivary gland sporozoites were approved by Emory University’s or the CDC’s IACUC and followed accordingly. All non-human primates used in this study were provided regular environmental enrichment opportunities consisting of daily feeding enrichment, provision of manipulanda, and physical enrichment. Subjects were regularly monitored for any behavioural signs of distress by YNPRC behaviour management personnel. Animals were trained using positive reinforcement to allow blood collections from the ear without sedation.Five healthy, male malaria-naïve Plasmodium cynomolgi B strain was selected for infections because the relapse pattern of this strain had been characterized previously [eviously , providieviously , needed P. cynomolgi B strain, ring-stage cryopreserved stocks maintained by the malaria branch at the CDC. Prior to the start of this study, the identity of these parasites was verified by Illumina 454 sequencing and comparison to the reported genome sequence of Tachibana et al. [Anopheles dirus, Anopheles gambiae and Anopheles stephensi mosquitoes were fed on the donor monkey using standard, IACUC-approved procedures after observance of male and female gametocytes in the blood. Successful infection of the mosquitoes was confirmed within a 7-day period post feeding via midgut dissections to monitor oocyst development and the progression of the infections with sporozoites detected in the salivary glands on days 11 and 12 after mosquitoes fed on infected blood. The salivary glands were freshly dissected from the infected mosquitoes and immediately processed by repeated passage through a 26G needle ten times to release sporozoites in RPMI-1640 culture media supplemented with 10 % fetal calf serum (FCS) on ice. The processed salivary gland and other mosquito debris were allowed to settle by gravity and the sporozoites in the supernatant were washed once by centrifugation at 1000×g in sterile culture medium with 10 % FCS. Sporozoite concentrations were determined by counting in a neubauer hemocytometer chamber using a light microscope at 400× magnification. Isolated sporozoites were re-suspended in sterile RPMI 1640 with 10 % FCS, and the macaque cohort infections were initiated by the intravenous inoculation of 2000 sporozoites per animal.To generate sporozoites, a donor rhesus macaque was infected with blood-stage parasites that were reconstituted from a et al. . LaboratBlood samples were collected daily from the monkey cohort from day −1 to 100 into EDTA-coated capillary tubes using standardized, IACUC-approved, ear-prick procedures. These samples were used to perform CBCs using an automatic cell analyzer  and reticulocyte enumeration using new methylene blue staining of thin blood smears. Clinical data were reviewed with parasitological data daily to monitor the clinical conditions of the animals and determine when specimens should be collected based on the criteria noted in Table Parasitaemia counts (parasites/µl) were determined daily via microscopy readings of thick and thin blood films stained with a modified Wrights–Giemsa using enumeration protocols recommended by the World Health Organization (WHO) . BrieflyPeripheral blood and bone marrow aspirate samples were collected prior to infection and during the infections Fig. a for immClinical definitions were based on reference values for rhesus macaques as defined at the YNPRC and other available literature . The norWHO criteria for the management of severe malaria were utilized as a guide to determine when animals were progressing towards severe disease and necessitated clinical and/or therapeutic intervention to ward off terminally severe outcomes . Animalsvs capillary blood collected daily) did not influence associations. A Wilcoxon matched-pairs rank sums test was performed to compare the primary and relapse infection parasitaemias. Both Graphpad Prism Version 6 and JMP Version 12 were used for statistical analysis.Non-parametric statistical approaches were used to assess statistical significance. A Friedman test was performed followed by Dunn’s Multiple Comparison post hoc analysis to identify significant changes in clinical parameters from pre-infection values over time and between pre-, primary, and relapse infection values. Spearman’s non-parametric correlation analysis was performed on clinical parameters using clinical values obtained from the defined experimental periods to ensure that different sample types . Data that were manually generated, e.g., clinical assessments, parasitaemias, animal handing, treatment, diet, snacks, and procedures were entered into the LIMS or electronic spreadsheets with double-data entry to increase accuracy. Data fields were constrained with limits or populated with pull-down lists of allowable values to further increase accuracy. All data were then transferred to internal repositories for validation and integration with other data. Standard operating procedures (SOPs) for all methods were collected and archived. Data derived from blood and bone marrow samples were associated with the appropriate animal and experimental metadata via a barcode. All data associated with this study  were backed-up locally as well as off-site. The dataset supporting the conclusions of this article is included with the article as Additional file P. cynomolgi and P. coatneyi in rhesus macaques at the CDC and the YNPRC [P. cynomolgi infections  consistent with previous literature  however, based on the four of five animals remaining in the study. A follow-up study will expand on these numbers. Contrary to a previous report stating that relapse parasitaemias decrease with subsequent relapses [The peak parasitaemia for the first relapse infections ranged from 846 to 11,584 parasites/µl, which was strikingly lower than the primary infection , a statistically significant decrease in haemoglobin concentration compared to pre-infection values did not coincide with the peak of parasitaemia during the acute, primary phase of the infection , and furthermore, severe thrombocytopaenia  was not observed  parasites/µl whereas the two severe animals that survived had parasitaemias of 309,365 and 379,140  parasites/µl during the primary infection , appeared to contribute to the lethality since this parasitaemia clearly distinguished this animal from the two non-severe (RSb14 and RIc14) and two severe (RMe14 and RFa14) clinical presentations. These phenotypes did not clearly stratify away from each other based on parasitological profiles alone, indicating that while parasitaemia may play a role in severe disease, it is not solely responsible for the observed differences in clinical presentation and other pathological processes may come into play. This is reminiscent of human malaria where individuals can progress to severe malaria regardless of parasitaemia [It is important to recognize that macaques infected with sitaemia –63. CuriP. cynomolgi B strain sporozoites inoculated, and based on this evidence, some investigators have used 1 × 106 sporozoites to generate early, frequent and uniform relapse patterns across individuals for screening anti-hypnozoite drugs [P. vivax infections with tropical strains of this parasite [Schmidt  demonstrte drugs . In contte drugs , 65, 200parasite , 66, 67.P. vivax, and also P. ovale, remain key challenges in today’s malaria eradication efforts, especially if asymptomatic carriers remain infectious to mosquitoes. Critically, the relative impact of primary versus relapse clinical malaria presentations has remained undefined in a controlled, experimental model system and direct evidence from human studies is not available because of the challenges distinguishing the two in human cases without a well-controlled study design [bona fide relapse, subsequent to blood-stage treatment and in the absence of re-infections, resulted in significantly lower parasitaemias compared to the primary infections and minimal, if any, changes in clinical parameters to indicate illness. This suggests that immunity, or potentially other undefined mechanisms, during the primary infection, led to controlled relapses characterized by reduced parasitaemias and lack of clinical complications. Whenever minor alterations in clinical parameters were observed during a relapse , as was the case for RMe14, these alterations resolved in a controlled, non-pathological manner, without the need for clinical support. The lack of clinical illness during relapses was not necessarily expected, especially in animals that only experienced a single primary blood-stage infection, since previous reports have concluded, through mathematical and statistical modelling of data collected from holo-endemic areas that relapses are responsible for up to 96 % of blood-stage P. vivax infections [Understanding, preventing and treating relapses caused by y design –71. Herefections . Thus, tfections , 74.P. cynomolgi were treated soon after infected RBCs were detected, and the parasitaemia of the first relapses appeared to be higher than subsequent relapses [In a previous study, rhesus macaques infected with relapses . The cliAs shown by this research team and others using this model system, relapse infections can be definitively produced under controlled experimental conditions , 32, in P. cynomolgi-rhesus macaque model is invaluable for malaria research, particularly with regard to the direct study of both primary and relapse infections and pathophysiological mechanisms. This study uniquely provides a day-to-day clinical perspective in haematological changes during longitudinal infections, and sets the stage for systems biology investigations based on data from a few individuals [bona fide relapses did not result in signs of clinical malaria. This was not necessarily expected and is important to consider when assessing both the clinical and epidemiological impact of relapses caused by P. vivax and P. ovale.The ividuals , 77. Her


Plasmodium falciparum infection is cerebral malaria, in which vascular leakage and cerebral swelling lead to coma and often death. P. falciparum produces a protein called histidine-rich protein II (HRPII) that accumulates to high levels in the bloodstream of patients and serves as a diagnostic and prognostic marker for falciparum malaria. Using a human cerebral microvascular endothelial barrier model, we previously found that HRPII activates the endothelial cell inflammasome, resulting in decreased integrity of tight junctions and increased endothelial barrier permeability. Here, we report that intravenous administration of HRPII induced blood-brain barrier leakage in uninfected mice. Furthermore, HRPII infusion in P. berghei-infected mice increased early mortality from experimental cerebral malaria. These data support the hypothesis that HRPII is a virulence factor that contributes to cerebral malaria by compromising the integrity of the blood-brain barrier.A devastating complication of  Plasmodium falciparum, which can cause complications such as severe anemia, respiratory distress and cerebral malaria (CM). CM manifests with a progression of symptoms from decreased consciousness to coma and death. Cerebral edema due to blood-brain barrier (BBB) compromise ultimately results in brain herniation and death [Malaria is a disease that afflicts several hundred million people each year. Most of the estimated 600,000 deaths  are due nd death .P. berghei ANKA serves as a small animal model for cerebral malaria. The pathology present in experimental cerebral malaria (ECM) is similar to that in human cerebral malaria (CM) with notable exceptions being limited sequestration of infected RBC and a more robust immune response enriched in leukocytes [Infection of mice with the rodent malaria parasite strain ukocytes –5. The bukocytes –9.P. falciparum but not by other malaria parasite species. It accumulates in the bloodstream [Histidine-rich protein II (HRPII) is a protein produced by odstream  and is todstream . This seodstream .In the current study, we evaluated the effect of HRPII infusion in mice. We find that HRPII causes cerebral vascular leakage in uninfected mice and increases the incidence of early death in a rodent malaria model. Blockade of inflammasome signaling with anti-IL1β antibody mitigates cerebral barrier compromise. These data support the hypothesis that HRPII contributes to the pathogenesis of cerebral malaria.in vivo studies along with an Armenian hamster IgG isotype control , at 300 μg/mouse. Dilutions were made in PBS.Armenian hamster anti- mouse IL-1β was purchased  and used for E. coli lysate using nickel-affinity chromatography as described (27). Protein was exchanged into 20 mM Tris, 500 mM NaCl, 50 mM imidazole and loaded on a 5 ml nickel column . After washing with 60 column volumes of 20 mM Tris, 10 mM NaCl, 0.1% Triton X-114 to remove residual LPS, the column was washed with 20 column volumes of loading buffer and eluted with loading buffer with 1 M imidazole. All preparations of HRPII were tested for residual LPS using a LAL endotoxin test ; levels administered to mice contained less than 5 EU/kg. Fully active preparations of the protein were used for experiments. Activity was measured using a Factor Xa assay [The coding sequence for the mature form of HRPII was cloned into pET-15b (Novagen) without a tag, expressed and purified from Xa assay . ProteinP. berghei ANKA parasites (105 parasites/100 μl) derived from stock mice  with parasitemias of <3%. All efforts were made to minimize animal suffering. We needed to use early death (6–9 days) as an endpoint rather than euthanization of ill-looking animals because the mice are capable of recovering from infection and it was important to distinguish those that recover from those that do not. Animals that survived the early period (30–70%) were euthanized for high parasitemia or sick appearance, using ketamine/xyazine overdose.Four-week old female C57BL/6 mice were purchased from Taconic. Animals were housed under pathogen-free conditions. All experiments were approved by and performed in compliance with Animal Studies guidelines at Washington University and Johns Hopkins University. Mice were given retro-orbital intravenous injections  approximately 12 hours prior to infection. The mice were infected by retro-orbital inoculation of Two doses of recombinant HRPII (200 μg in 100 ul) were given 24 hours apart by retro-orbital injection to 4-week old female C57BL/6J mice from Jackson Labs. 48 hours post initial injection, the mice were evaluated for sodium fluorescein extravasation as previously described . In someAll data were analyzed using Graph Pad Prism 6.0 . A p<0.05 was designated as significant. Pair-wise comparisons were analyzed by two-tailed t-test. Log rank test was used to compare survival between the groups in the Kaplan-Meier survival curves.in vitro [in vivo, uninfected mice were administered two 200 μg doses of HRPII 24 hours apart by intravenous (IV) injection, and fluorescein extravasation was measured in the brain parenchyma . The parasitemia of mice dying from cerebral malaria-like symptoms was low, as expected, and importantly, no differences in parasitemia were observed between mice infused with HRPII or control protein.We next determined whether the compromise in vascular integrity observed with purified HRPII had clinical consequences during malaria infection in mice. We infused 6-week old female C57BL/6 mice with 50 μg of BSA or HRPII prior to infection with 2 x10 malaria ,15 definPlasmodium falciparum infection is a hallmark of CM. Our previous studies identified HRPII as a parasite virulence factor that activates host endothelial innate immunity through an inflammasome-mediated pathway. This causes redistribution of endothelial junctional proteins, and increased BBB permeability.BBB integrity disruption during in vivo, resulting in leakage of fluorescein into the brain parenchyma of uninfected mice as well as an exacerbation of experimental CM when the protein is infused into mice prior to infection. Mouse endothelial cells are less sensitive to HRPII than human cells (not shown) and achievable plasma levels of HRPII are considerably lower than those seen in human patients with cerebral malaria, where levels of 1–100 μg/ml have been reported [P. berghei pathogenesis were measured.In the current work, we found that HRPII is active reported . These lP. falciparum infection [Given the long half-life of HRPII in the bloodstream of malaria parasites , ounfection . WhetherBlocking HRPII action has potential as an adjunctive therapy for falciparum malaria. Anti-IL-1β antibody was able to mitigate the effect of HRPII on vascular leakage in our mouse infusion studies. Drugs targeting IL-1β are already in clinical use, and these or other inhibitors that work elsewhere in the HRPII pro-inflammatory pathway could be considered for the treatment of cerebral malaria.While the current work is focused on the brain, it will be interesting to see if other vascular beds are sensitive to HRPII, since falciparum malaria is a multi-organ disease. The long half-life of HRPII in the human bloodstream makes this molecule a candidate to explain the failure of antimalarial drugs to reverse cerebral malaria symptoms in some cases, even after parasite clearance. Similarly, it could offer an explanation for post-malaria syndrome symptomatology and for the persistent endothelial activation that has been observed after falciparum malaria has been treated.


Plasmodium vivax. We hypothesize that malaria infection may partially protect against dengue – in particular falciparum malaria against severe dengue – and that this inter-species cross-protection may explain the near absence of severe dengue from the Sub-Saharan region and parts of South Asia until recently. We speculate that malaria infection elicits cross-reactive antibodies or other immune responses that infer cross-protection, or at least partial cross-protection, against symptomatic and severe dengue. Plasmodia have been shown to give rise to polyclonal B-cell activation and to heterophilic antibodies, while some anti-dengue IgM tests have high degree of cross-reactivity with sera from malaria patients. In the following, the historical evolution of falciparum malaria and dengue is briefly reviewed, and we explore early evidence of subclinical dengue in high-transmission malaria areas as well as conflicting reports on severity of co-morbidity. We also discuss examples of other interspecies interactions.The global malaria burden, including falciparum malaria, has been reduced by 50% since 2000, though less so in Sub-Saharan Africa. Regional malaria elimination campaigns beginning in the 1940s, up-scaled in the 1950s, succeeded in the 1970s in eliminating malaria from Europe, North America, the Caribbean (except Haiti), and parts of Asia and South- and Central America. Dengue has grown dramatically throughout the pantropical regions since the 1950s, first in Southeast Asia in the form of large-scale epidemics including severe dengue, though mostly sparing Sub-Saharan Africa. Globally, the WHO estimates 50 million dengue infections every year, while others estimate almost 400 million infections, including 100 million clinical cases. Curiously, despite wide geographic overlap between malaria and dengue-endemic areas, published reports of co-infections have been scarce until recently. Superimposed acute dengue infection might be expected to result in more severe combined disease because both pathogens can induce shock and hemorrhage. However, a recent review found no reports on more severe morbidity or higher mortality associated with co-infections. Cases of severe dual infections have almost exclusively been reported from South America, and predominantly in persons infected by  Plasmodium falciparum malaria parasites probably emerged as a human pathogen from its enzootic origin in West Africa about 5,000–10,000 years ago ; some suggest an African origin, others an Asian, given the presence of sylvatic (“jungle”) viruses in both regions . Irrespe aegypti  and hist aegypti .Ae. aegypti, while travel and trade have helped widen the global range of both vector and virus  in a given population. Dengue epidemics involving severe disease with mortality rates as high as 10% were first noted in Manila and Bangkok in 1954 , but werWhereas the Pacific islands experienced their first outbreaks with severe dengue in the 1970s, the phenomenon was not reported in the Americas before 1981, when Cuba reported a large-scale epidemic with tens of thousands of severe dengue cases . The subAe. aegypti in the coastal area of Kenya. In addition, 47% of the population presented with hemagglutinin inhibiting (HI) dengue antibodies, although no outbreaks of dengue had been reported. In 1977, Fagbami et al. found that 45% of tested adults in Nigeria were seropositive for anti-DENV-2 antibodies, as evidenced by neutralizing antibodies .Recent reviews have cataloged multiple small-scale outbreaks of the 1980s and 1990s, across Sub-Saharan Africa . InteresIt is generally argued that dengue and severe dengue have been subject to under-diagnosis in Africa given a lack of index of suspicion and little access to laboratory diagnostics . HoweverSeveral factors have been shown to modulate the clinical response to DENV infection. Notable risk factors for severe manifestations include previous heterotypic DENV infection; viral virulence, co-morbidities such as diabetes and hypertension; as well as host genetic factors .in vitro) to enhance viral uptake and proliferation in target cells, a mechanism known as antibody dependent enhancement (ADE) of infection with a second dengue serotype. Notably, recent studies suggest that IgG DENV-antibodies may also be linked to thrombocytopenia as observed in severe dengue . P. vivax was identified in the majority of cases (∼80%) and that only 50% of the dual infected reported previous malaria. Unfortunately, the report did not stratify between P. vivax or P. falciparum co-infections, nor did it report on parasitaemia levels.Lack of reporting of dual infection in the field could result from testing positive for either malaria or dengue infection with no further testing being done, given low index of suspicion for co-infections . Yet, some argue that severity could also prompt testing for dual disease, and thereby potentially bias reporting. Notably, studies that have actively investigated the clinical picture of mono-infections as compared to that of dengue and malaria co-infections disagree with respect to observed differences in severity levels. P. vivax. In India, P. falciparum infections. Notably, lower parasitic counts were observed among co-infected than for mono-infected patient, although reported as non-significant. In line with these findings, Ahmad reported that the presentation and severity of co-infections appeared “to be milder and outcomes comparable (if not better) to mono-infections” during a dengue outbreak in Uttarakhand, India. While co-infections occurred with both P. falciparum and P. vivax these were not clearly distinguished in the report.In contrast, P. falciparum parasitaemia, suggesting previous exposure, and dengue IgM antibodies in 3%, indicating probable acute co-infection. While co-infection was indicated, co-morbidity or increased severity was not. None of the children appeared to have severe disease; but IgM dengue positive children had (non-significantly) higher levels of P. falciparum parasitaemia, which could indicate they were only partially immune to falciparum malaria at the time of DENV infection. Unfortunately, levels of malaria antibodies were not reported. Specificity of the anti-dengue Ig tests used was 97% for multisite pooled sera, though cross-reactivity with other flavivira was high .Malaria infection gives rise to polyclonal B-cell activation  and to hRepeated exposure to falciparum malaria is followed by development of protective antibodies, decline of pro-inflammatory host-responses  and decrProof of concept of cross-protection from malaria to dengue in man would require exposing malaria immune humans to dengue in controlled experiments. As reviewed by P. falciparum infection may protect partly against severe Ebola outcomes , with the reduction in incidence of malaria , suggesting that declining malaria exposure due to successful control programs or other environmental factors may have inadvertently led to the cessation of protection against clinically apparent and severe dengue infections, especially in Sub-Saharan Africa. However, individual-level studies would be the best way to determine if co-protection exists between falciparum malaria and dengue. Available individual-level studies reviewed here are only suggestive and interpretation hampered by non-specificity of used tests. Also, they present a risk of bias in favor of our hypothesis, in that more severe patients are more likely to be tested for several pathogens and subsequently reported, and vice versa that lack of reporting of dual infection in the field could result from testing positive for either malaria or dengue infection and then no further testing being done.In summary, we conclude that at the population level the evidence of interaction between dengue and malaria is perhaps already there: when comparing time trends in the two diseases at continent level, a suggestive pattern emerges. We have discussed the striking coincidence of the emergence of dengue in recent years  of cross-protection. Other modern in vitro technologies including epigenetic silencing or activation of regulatory genes implicated in immunity and susceptibility of disease (Evidently, more research is required to investigate whether there is a causal link between declining falciparum malaria and increased dengue in recent years. We suggest  disease  or devel disease , and stu disease  could be disease . These s disease .Finally, we believe it is important to broaden considerations across pathogens and diseases in order to explain the striking associations in temporal patterns of malaria and dengue globally observed since the 1950s and in Africa, specifically, after the Millennium. More broadly, we suggest that considering non-specific effects of inter-species interaction and interdependences may be a critical aspect of recognizing possible outcomes beyond those of the target pathogen and to understand the broader consequences of global health intervention programs designed to eliminate a given disease.Table 1 and to the Supplementary Material, and contributed to all sections and versions of the manuscript. LS contributed in particular to the sections on historic epidemiology and interspecies interaction and to other sections and versions of the manuscript. All authors read and approved the submitted version.IB conceived the hypothesis of potential interaction between malaria and dengue, and drafted the first version of the manuscript, and wrote the subsequent versions in close collaboration with KS and LS. KS contributed in particular to the sections of dengue and malaria epidemiology mapping, and to The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.