Plasmodium knowlesi, in addition to many potential parasites, have urged the interest of scientific community. A broad spectrum of protozoal parasites frequently affects the respiratory system, particularly the lungs. The diagnosis of parasitic diseases of airway is challenging due to their wide varieties of clinical and roentgenographic presentations. So detailed interrogations of travel history to endemic areas are critical for clinicians or pulmonologists to manage this entity. The migrating adult worms can cause mechanical airway obstruction, while the larvae can cause airway inflammation. This paper provides a comprehensive review of both protozoal and helminthic infestations that affect the airway system, particularly the lungs, including clinical and roentgenographic presentations, diagnostic tests, and therapeutic approaches.Parasitic infestations demonstrated a decline in the past decade as a result of better hygiene practices and improved socioeconomic conditions. Nevertheless, global immigration, increased numbers of the immunocompromised people, international traveling, global warming, and rapid urbanization of the cities have increased the susceptibility of the world population to parasitic diseases. A number of new human parasites, such as  Toxoplasma gondii pneumonia is observed most frequently in acquired-immunodeficiency-syndrome (AIDS) patients. Pulmonary strongyloidiasis is identified in patients with chemotherapy or glucocorticoids treatment and is endemic in the Southeastern USA, whereas Ascaris and hookworm infestations may be present with eosinophilia and pulmonary infiltrates during the larval migration stage [ Entamoeba histolytica, Paragonimus, and Dirofilaria lung involvement are less common [Protozoal and helminthic parasitic pneumonias and lung involvement are common in the tropics  with fewon stage . Neverthon stage . In 1932on stage , whereas Plasmodium falciparum, Plasmodium malariae, Plasmodium vivax, and Plasmodium ovale, and the protozoa of the genus Plasmodium cause Malaria and are primarily transmitted by the bite of an infected female Anopheles mosquito to infect humans [ Plasmodium falciparum [ Plasmodium vivax, one of the two forms  of relapsing malaria to infect humans, and is the most prevalent in Southeast Asia and South America, has ability to become dormant in the liver (“hypnozoite”) and is able to be reactivated after months or years contributing to an attack of intraerythrocytic stage malaria despite the absence of mosquito bites [ Plasmodium falciparum, and the cycle is able to develop clinical symptoms [ Plasmodium falciparum [ Plasmodium falciparum, respectively, in the mosquito's midgut [ Plasmodium falciparum before transforming to oocysts and sporozoites in the mosquito's salivary gland before transmission of the sporozoite to humans by mosquito biting [ Plasmodium falciparum, while the longer ones are observed in Plasmodium malariae [ Plasmodium falciparum in the microvasculature of the organs and is quantified by measurement of Plasmodium falciparum specific histidine-rich protein 2 (PfHRP2) using a quantitative antigen-capture enzyme-linked immunosorbent assay [The four types of malarial parasites aret humans . The malt humans . This stlciparum . In caseto bites , 7. The to bites . This cysymptoms . In anotsymptoms . This stlciparum . Human llciparum . After ms midgut . The dipo biting . The malo biting . In mostnt assay . Gas excnt assay .Plasmodium falciparum, Plasmodium vivax, and Plasmodium ovale) and on every third day with “quartan” parasite  [ Plasmodium falciparum may result in severe anemia [ Plasmodium malariae [ Plasmodium falciparum may cause more severe disease in mother and may lead to premature delivery or delivery of a low-birth-weight baby [ Plasmodium vivax can cause rupture of the spleen [ Plasmodium falciparum have been introduced [ Plasmodium vivax malaria demonstrated diffuse bilateral alveolar opacities which indicated acute respiratory distress syndrome [ Plasmodium vivax malaria were also reported in India and one case was demonstrated with bilateral perihilar infiltrates, one case with bilateral diffuse extensive opacities, and another case with bilateral basal ground glass opacities on chest roentgenograms [ Plasmodium berghei ANKA strain infection in a mice model [ in vitro studies, Plasmodium falciparum merozoite proteins could increase pulmonary endothelial permeability, whereas Plasmodium falciparum infected-red blood cells did not demonstrate the same properties indicating that the effects of the malaria parasites on the pulmonary endothelium probably mediated the activity of Src-family kinases [ Plasmodium berghei K173 demonstrated that proteins and inflammatory cells mainly CD4+ and CD8+ lymphocytes, monocytes, and neutrophils accumulated in the lungs of infected mice [α, interferon-γ, CXCL10 and CXCL11, as well as neutrophil and monocyte chemo-attractant chemokines  in the lungs [ Plasmodium falciparum malaria, ARDS patients with Plasmodium vivax, Plasmodium ovale, and Plasmodium knowlesi were also reported [ Plasmodium falciparum and could be partially attributed the resetting and sequestration of parasite-infected red blood cells in the pulmonary microcirculation [ Plasmodium vivax malaria and principally could be due to dysregulation of cytokine production [ Plasmodium knowlesi indicates that parasite-specific effects increase pulmonary capillary permeability but could contribute to hypoxemia and metabolic acidosis [ Plasmodium falciparum infections and those rare cases of life-threatening malaria caused by Plasmodium vivax, Plasmodium malariae, and Plasmodium ovale [ Plasmodium falciparum chloroquine-resistance transporter (PfCRT) gene is responsible for chloroquine-resistant falciparum malaria [ Plasmodium species in both children and adults [ Plasmodium species and chloroquine-resistant Plasmodium vivax in both children and adults [In patients with uncomplicated malaria, the classical (but infrequently observed) malaria attack lasts 6–10 hours that consists of a cold stage , a hot stage , and a sweating stage  . Classicalariae) . More coalariae) . The phyalariae) . In patialariae) . Recurree anemia . Nephrotmalariae . Hyperremalariae . Plasmodght baby . Neurologht baby . On raree spleen . The mose spleen . Additioe spleen . Human utroduced . In sevetroduced . Sanklectroduced . Chest rtroduced . Chest rtroduced . Chest rsyndrome . Three cenograms . Pulmonaenograms . The alvenograms . In manyenograms . This isenograms . There ienograms . The majenograms . Mononucenograms . Pulmonaenograms . A recence model . About 6ce model . There ice model . Increasce model . In in v kinases . Increas kinases . A studyted mice . Van denhe lungs . Pulmonahe lungs . Despitereported . The greculation . Heavy poduction . Increasacidosis . Intrave malaria , whereas malaria . Oral ar malaria , 23. Add malaria . The WHOd adults . Oral ard adults . Hence, d adults . Treatmed adults . It is dd adults . Dexametd adults .Entamoeba histolytica, a well-recognized pathogenic amoeba, is associated with both intestinal and extraintestinal infections [ Entamoeba histolytica, occurs mainly by the extension from the amoebic liver abscess [ Entamoeba histolytica and Entamoeba dispar, respectively, have been established [ Entamoeba histolytica is identified with ingested red blood cells (erythrophagocytosis) [ Entamoeba histolytica-exposed persons develops clinical disease is not fully understood [ Entamoeba histolytica and its virulence is not yet known [ Entamoeba histolytica as other two non-pathogenic species found in humans (Entamoeba dispar and Entamoeba moshkovskii) are morphologically indistinguishable but can be rapidly, accurately, and effectively diagnosed by a single-round PCR [ Entamoeba histolytica, enzyme-linked immunosorbent assay (ELISA), indirect fluorescent antibody test (IFAT), and indirect hemagglutination test (IHA) [ Entamoeba histolytica can be identified in sputum or pleural specimen [ Entamoeba cysts [fections . Both cyfections . Cysts afections . Humans fections . Excystafections . The trofections . Due to fections . In contfections . Surprisfections . In somefections . Pulmona abscess , 29. Theablished . Entamoecytosis) . Transmicytosis) . The immderstood . The effet known . The preound PCR , 31, 32.st (IHA) . Active specimen . Diloxanba cysts . Lactofeba cysts . Identifba cysts . Leishmania donovani and Leishmania chagasi or infantum [ Phlebotomus, a type of sand fly [ Leishmania amastigotes can be identified in pulmonary septa, alveoli, and the BAL fluid [ Leishmania infantum and HIV-1 that may affect the parasite control in visceral leishmaniasis coinfected patients [ Leishmania species are indicated in the pretransplantation screening from endemic areas [Pulmonaryor visceral leishmaniasis, also called “Kala azar” is caused byinfantum , is transand fly . LeishmaAL fluid , 40. PleAL fluid , 40. TheAL fluid  indicatiAL fluid . Accordipatients . In comppatients , 43. On patients , 45. Henic areas . Immune ic areas . Pentavaic areas . A previic areas .Trypanosoma brucei gambiense” [Trypanosoma brucei rhodesie” [Trypanosoma cruzi” which was discovered by Carlos Chagas in 1909 [ Trypanosoma cruzi without affecting the left ventricle [Trypanosoma cruzi drugs [Human African trypanosomiasis (HAT) or sleeping sickness is caused by an extracellularly protozoan parasite, called “mbiense” , “Trypanhodesie” , and “Tr in 1909  and is e in 1909 . HAT con in 1909 , 53. In  in 1909 . Thicken in 1909 . These b in 1909 . Most in in 1909 . These p in 1909 . Pulmona in 1909 . By the  in 1909 . Most Me in 1909  and coulentricle . Howeverentricle . A previentricle . In animentricle , 57. A rzi drugs .Toxocara larval migrans caused by Toxocara canis, a parasite in dogs' intestine, and Toxocara cati, a parasite in cats' intestine, infected intermediate host, humans, by ingestion of these embryonated Toxocara eggs which hatch into infective larvae in the human intestine [ Toxocara Canis, and increased total serum IgE level [ Toxocara excretory-secretory antigens by Western-blotting method has been reported for diagnosis [ Toxocara eggs or larvae cannot be identified in the feces since human is not the definitive host [ Toxocara larval migrans may be spontaneous resolution; therefore, mild to moderate symptomatic patients need not any treatment [ Toxocara larval migrans can be treated with diethylcarbamazine  [ntestine . The infntestine . Granulontestine . Pulmonantestine . Clinicantestine . Some cantestine –61. ChesgE level , 63. AbogE level . IdentifgE level  and Toxoiagnosis . Neverthiagnosis , 66. Tox21 days) , mebenda21 days) , or albe21 days) . ExacerbToxoplasma gondii” which are primarily carried by cats, is causal microorganism [ Toxoplasma gondii tachyzoites in sputum smears [ Toxoplasma gondii infection due to ability to increase the antibody levels without active disease [A celled protozoan parasite, called “organism . Humans organism . The cliorganism  which isorganism . Pulmonaorganism . Pulmonaorganism . Neverthorganism . Early porganism . Diagnosorganism . Sputum m smears . Serodia disease . A real- disease . Toxopla disease . Babesia divergens and Babesia microti [ Ixodes scapularis, or by contaminated blood transfusion [ Plasmodium [This disease is caused by haemoprotozoan parasites, microti . Humans nsfusion . The parasmodium . The symasmodium . Patientasmodium . Giemsa-asmodium . Treatmeasmodium , 80. Wuchereria bancrofti and Brugia malayi [ Dirofilaria immitis antigens has been demonstrated in patients with TPE [ Wuchereria bancrofti, had been demonstrated in the lungs, liver, and lymph nodes of the patients with TPE [ Wuchereria or Brugia antigens than with Dirofilaria antigen [ Wuchereria bancrofti revealed maximal positive results compared with Dirofilaria immitis and Dirofilaria repens [ Wuchereria bancrofti in the lymphatic vessels of the spermatic cord of the patients with TPE is evidenced by ultrasound examination [This syndrome results from immunological hyperresponsiveness to human filarial parasites,a malayi . Tropicaa malayi , 82. Clia malayi . Chest ra malayi . Additioa malayi –88, but a malayi . Some pra malayi . At leasa malayi , but onla malayi , whereasa malayi , 92. A pwith TPE . Microfiwith TPE –96 but awith TPE . Filariawith TPE . Periphe antigen . This in antigen . Leukocya repens . Demonstmination  and biopmination . There imination  and roenmination , 103 aftmination . The usemination , 106. Dirofilaria immitis [ Dirofilaria repens and Dirofilaria tenuis, are known to infect humans [Aedes, Culex, or Anopheles) [Human pulmonary dirofilariasis, a disease of middle-age adults , is caus immitis –112 Othet humans . These ot humans –115 to ht humans  by infecopheles) , with a opheles) . Majoritopheles) , 118. Thopheles) , as noteopheles) . Patholoopheles) . Clinicaopheles) –121. At opheles) , 122. Onopheles) . Definitopheles) , 125. Weopheles) . Some suopheles) . Strongyloides stercoralis which is commonly found in many tropical and subtropical areas, has occasionally been reported as the cause of pulmonary disease [ Strongyloides stercoralis [ Strongyloides stercoralis [ Strongyloides stercoralis detected in the stool despite the rarity of clinically significant strongyloides infection in HIV-infected/AIDS patients [Very few parasitic diseases have been reported to cause pneumonia in HIV-infected/AIDS patients . A helmi disease . The pre disease . Althoug disease . There h disease . In a prrcoralis , whereasrcoralis . The parrcoralis . Eggs arrcoralis . The rharcoralis . These lrcoralis . This corcoralis . The rharcoralis . In dirercoralis . In indircoralis . These rrcoralis . These frcoralis . In the rcoralis . Followircoralis . During rcoralis . The patrcoralis . These prcoralis . Most parcoralis . The defrcoralis . Previourcoralis , 135. A Ascaris lumbricoides is the most common intestinal helminthic infection [ Ascaris lumbricoides can produce both specific and polyclonal IgE [nfection . Both fenfection . Infectinfection . The eggnfection . The secnfection . The founfection . It takenfection . The mignfection . This manfection . Ascarisonal IgE . Elevational IgE . Symptomonal IgE , 140. Thonal IgE , 140 to onal IgE . Chest ronal IgE . MebendaAncylostoma duodenale can live only one year [ Ancylostoma duodenale produces 10,000 to 30,000 eggs per day [ Ancylostoma duodenale larvae can enter the human host via the oral route in addition to the skin and it can reach pulmonary circulation through the lymphatics and venules [ Ancylostoma duodenale larvae can developmentally get arrested in the intestine or muscle and restart development when environmental conditions become favorable [one year , 144. Fe per day , 144. Ma per day , 144. An venules . Ancylosavorable . Bronchiavorable , 144. Puavorable , 144. Hoavorable , 144 andavorable . During avorable . Both alavorable . Pyranteavorable . A previavorable .Necator americanus larvae can infect human only through the skin [ Ancylostoma duodenale [ Necator americanus are the same as the drugs of choice for treatment of Ancylostoma duodenale [the skin . The laruodenale . The intuodenale . Bronchiuodenale . Drugs ouodenale . Paragonimus species such as Paragonimus westermani which is the main species in humans, Paragonimus mexicanus, Paragonimus africanus, Paragonimus miyazakii, Paragonimus philippinensis, Paragonimus kellicotti, Paragonimus skrjabini, Paragonimus heterotremus, and Paragonimus uterobilateralis [ Paragonimus species when they ingest raw crayfishes or crabs infected with infective metacercariae [ Paragonimus ova-positive patients, particularly cough and hemoptysis [ Paragonimus cystic lesions [ Paragonimus ova-positive sputum in 55.6% to 72% of sputum specimens of patients with pulmonary paragonimiasis [ Paragonimus ova may be demonstrated in the centrifuged deposit of pleural fluid in approximately 10% of cases presented with pleuropulmonary paragonimiasis [ Paragonimus ova are identified in the fecal samples of the patients with pulmonary paragonimiasis in approximately 65.1% by AMS III concentration technique [ Paragonimus westermani adult excretory-secretory products are composed of cysteine proteases which are involved in immunological reactions during parasitic infection [ Paragonimus heterotremus had accuracy, sensitivity, specificity, and positive and negative predictive values of 97.6%, 100%, 96.9%, 90%, and 100%, respectively [ Paragonimus ova, adult or immature worm, eosinophils, inflammatory cells, and Charcot-Leyden crystals [In Asia, nearly 20 million people are infected withateralis –149. Parateralis . Humans ercariae . The incercariae . The shoercariae . The parercariae . Adult wercariae . The eggercariae . The mirercariae . The cerercariae . Humans ercariae . The infercariae . The migercariae . Pulmonaercariae  that is ercariae , 152. Geercariae . The chrercariae . Generalercariae . Cough, moptysis . In chilmoptysis . This symoptysis . There imoptysis . Presencmoptysis . Both admoptysis . A recenmoptysis . Therefomoptysis . Pneumotmoptysis , 154. Chmoptysis , 155. Thmoptysis . Neverthmoptysis  and may moptysis –158. Permoptysis . Oloyedemoptysis . The maimoptysis . Compute lesions . The par lesions . Endobro lesions . Extrapu lesions . In adul lesions . Previounimiasis . A studynimiasis . Neverthnimiasis . Paragonnimiasis . Pleuralnimiasis . Examinanimiasis . Paragonechnique . Peripheechnique , 154. Panfection , 161. Manfection . While anfection . This tenfection . The ELInfection . Neverthnfection . A rapidnfection . Immunogectively . In caseectively . The meaectively . Excisioectively . Patholocrystals . Paragoncrystals ), triclacrystals , 165. Cocrystals .Schistosoma species that cause human disease are Schistosoma hematobium, Schistosoma japonicum, and Schistosoma mansoni [Schistosoma japonicum and Schistosoma mansoni) or in urine (Schistosoma hematobium) [Schistosoma japonicum and Schistosoma mansoni) and the urinary bladder vesicle beds (Schistosoma hematobium) [ Schistosoma involves two hosts: humans and snails [ Schistosoma mansoni and Schistosoma japonicum and the urinary vesicle beds in the case of Schistosoma hematobium [ mansoni . The schatobium) . The infatobium) . The lifd snails . The eggd snails , 166. Thd snails , 166. Pematobium . Acute mmatobium , 166–168 Schistosoma species can reach almost 100% [ Schistosoma japonicum and Schistosoma mansoni [ Schistosoma hematobium infection, the final habitat of the adult fluke is in the perivesical plexus, in which the eggs were laid by the mature flukes [ Schistosoma mansoni and Schistosoma japonicum, the eggs are swept with the portal blood flow and are lodged in the venules of the liver, creating a granuloma around the eggs, which finally causes periportal hepatic fibrosis and portal hypertension [ Schistosoma mansoni eggs in the lungs without evidence of hepatic fibrosis has been documented [ Schistosoma hematobium or Schistosoma japonicum causes much less cor pulmonale compared to Schistosoma mansoni [ Schistosoma hematobium is identified in pulmonary beds at significant percentages [The acute form is presented with dry cough, wheezing, shortness of breath, chill, fever, headache, malaise, weight loss, abdominal pain, diarrhea, urticarial, marked eosinophilia, arthralgia, myalgia , 169, anost 100% . This teost 100% . Howeverost 100% . A good ost 100% . Hepatos mansoni . In casee flukes . The ecte flukes . In casertension . Portal rtension . Demonstcumented . The eggcumented , 170. Lucumented . The eggcumented . The clicumented . It is e mansoni  althoughcentages . In chrocentages . Diagnoscentages . Bronchocentages . The opecentages . PCR tescentages . Both accentages . A shortcentages . A patiecentages . Echinococcus multilocularis, Echinococcus granulosus, Echinococcus vogeli, and Echinococcus oligarthrus [ Echinococcus multilocularis causes alveolar or pulmonary echinococcosis (AE), Echinococcus granulosus causes cystic echinococcosis, Echinococcus vogeli causes polycystic echinococcosis, and Echinococcus oligarthrus is an extremely rare cause of human echinococcosis [ Echinococcus multilocularis resides in the small bowel of the definitive hosts, foxes, dogs, cats, coyotes, and wolves [ Echinococcus granulosus, with the following differences: the definitive hosts are dogs and other canidae and the intermediate hosts are sheep, goats, swine, and so forth [ Echinococcus vogeli, the definitive hosts are bush dogs and dogs, the intermediate hosts are rodents, and the larval stage  develops both externally and internally, resulting in multiple vesicles [ Echinococcus oligarthrus, the life cycle involves wild felids as definitive hosts and rodents as intermediate hosts [ Echinococcus granulosus resides mainly in the small gut of the dogs [ Echinococcus granulosus antigens have preferable sensitivity and specificity for the diagnosis of AE [Human hydatid disease is caused bygarthrus , 177. Ecd wolves , 177. Inte hosts . The aduthe dogs . The intthe dogs . Clinicathe dogs . Rupturethe dogs , 180 andthe dogs . Ruptureis of AE . A serolis of AE . The mosis of AE . Chest ris of AE . Air-fluis of AE . Unusualis of AE . Treatmeis of AE . The treis of AE  but shouis of AE , 189. Trichinella spiralis [ Trichinella [Trichinella antibodies using excretory-secretory antigens [Trichinella IgG antibodies [ Trichinella spiralis infection; a definite diagnosis can be performed by muscle biopsy (preferably deltoid muscle) [The most important species that infects humans isspiralis . Humans chinella , 192. Thchinella . Fertilichinella . The larchinella . Clinicachinella , 195. Thchinella . Dyspneaantigens  or anti- muscle) . The alt muscle) . Symptom muscle) , 192. Dirofilaria ursi, Dirofilaria subdermata, Dipetalonema species, Loa Loa, Thelazia callipaeda, Babesia duncani, Babesia venato rum, Babesia-liked organisms, Babesia divergens-liked organism, Leishmania braziliensis, Leishmania major, Plasmodium knowlesi, Plasmodium simium, Onchocerca gutturosa, Onchocerca cervicalis, Onchocerca jakutensis, Onchocerca dewittei japonica, Onchocerca lupi [ Diphyllobothrium dendriticum [Macaca fascicularis) are believed to be the primary hosts of Plasmodium knowlesi [ Anopheles latens, a mosquito species that feeds on both themonkey natural host as well as on humans [ Plasmodium knowlesi, confirmed by PCR, has been reported from Thailand [ Trypanosoma cruzi-contaminated guava, sugar cane or acai' juices have been reported in South America [A number of new human parasites have urged the interest of scientific community in addition to many zoonotic potential parasites . Potentirca lupi , 198, Didriticum , and so knowlesi , identifn humans . A singl America . Leishmania, malaria, Schistosoma, Trypanosoma, Toxocara, or amoebiasis. Exposure to cats or dogs may indicate Toxocara or Ancylostoma infection. Multiple organ involvement may indicate Churg-Strauss syndrome. Clinical assessment must exclude other interstitial lung diseases and both primary and secondary lung malignancies. Education programs should be carried out in endemic areas for more awareness of the risk of oral transmission of Trypanosoma cruzi. Control measures are certainly not enough to stop the spreading of visceral leishmaniasis since global changes are accelerating this process. The real impact of global changes on the ecoepidemiology of the leishmaniasis in both traditional and nontraditional endemic areas might be unpredictable. Identification of priorities for society's scientific resources that are beneficial for global ecosystem integrity and human health and well-being at this complex intersection of humans, wildlife, domestic animals, and pathogens is difficult, but this problem can be solved by encouraging proactive exploration and assessment of potentially major influences on parasite flow from domestic animals and wildlife to humans, particularly environmental and climate changes, human intrusion into domestic and wildlife habitats, and other potential causes of ecosystem disruption. In arthropod or vector-borne parasite control, monitoring environmental and climatic changes remain a significant issue to be considered since these factors may affect both ecology and behavior of arthropod vectors. It is recommended that the national public health surveillance services should perform systematic entomological surveys in vector-free areas that are at risk of introduction of vectors through human or animal populations. Summary of parasitic diseases, chest roentgenographic features, and chemotherapeutic agents is demonstrated in Investigation of travel history is significant as this may indicate parasitic infection such as tropical eosinophilia,


Severe falciparum malaria is commonly complicated by metabolic acidosis. Together with lactic acid (LA), other previously unmeasured acids have been implicated in the pathogenesis of falciparum malaria.In this prospective study, we characterised organic acids in adults with severe falciparum malaria in India and Bangladesh. Liquid chromatography-mass spectrometry was used to measure organic acids in plasma and urine. Patients were followed until recovery or death.P<0.05). In urine, concentrations of methylmalonic, ethylmalonic and α-ketoglutaric acids were also elevated. Multivariate logistic regression showed that plasma HPLA was a strong independent predictor of death , comparable to LA  (combined area under the receiver operating characteristic curve 0.81).Patients with severe malaria (n=138), uncomplicated malaria (n=102), sepsis (n=32) and febrile encephalopathy (n=35) were included. Strong ion gap (mean±SD) was elevated in severe malaria (8.2 mEq/L±4.5) and severe sepsis (8.6 mEq/L±7.7) compared with uncomplicated malaria (6.0 mEq/L±5.1) and encephalopathy (6.6 mEq/L±4.7). Compared with uncomplicated malaria, severe malaria was characterised by elevated plasma LA, hydroxyphenyllactic acid (HPLA), α-hydroxybutyric acid and β-hydroxybutyric acid  contains supplementary material, which is available to authorized users. Severe falciparum malaria remains a leading cause of mortality in the tropics. The case fatality rate in adult patients remains between 15 % and 40 % in resource-constrained hospital settings, and it is 11 % in intensive care units in resource-rich settings . A betteThe degree of metabolic acidosis in severe malaria is a strong prognosticator for a fatal outcome. Acidosis is associated with hyperlactataemia, and lactic acid (LA) has therefore been considered a major contributor to hydrogen ion accumulation , 5. In cUsing the modified physicochemical approach of Stewart, the presence of an unmeasured anion load was detected in patients with severe malaria , 16. TheUsing a recently developed liquid chromatography-mass spectrometry (LC-MS) assay , we quanPlasmodium falciparum parasites visualised on a peripheral blood film, and severe malaria was distinguished from uncomplicated malaria by the presence of one or more markers of severity according to modified World Health Organisation (WHO) criteria as defined by Tran et al.  + [K+] + [Ca2+] + [Mg2+] − [Cl−] − [lactate−], with all concentrations expressed in milliequivalents per litre. The effective strong ion difference (SIDeff) was calculated by inclusion of the charges contributed by HCO3−, albumin and inorganic phosphate, using calculations described previously  2.1–7.0), compared with 4.2 % in patients with bacterial sepsis (IQR 2.0–15.6). The median proportion of SIG attributable to HPLA alone was 0.13 % in severe malaria (IQR 0.09–0.21), and 0.08 % in sepsis (IQR 0.04–0.18); these contributions were significantly different  and β-HBA . Weaker correlations were seen between bilirubin and all four acids and between the parasite biomass marker PfHRP2 and all four acids. GCS score correlated inversely with all four acids. The complete correlation matrix is provided in Additional file Plasma creatinine was correlated with HPLA . In the model of plasma acids in 138 patients (39 of whom died), HPLA and LA had independent prognostic value, with the combined model providing an AUROCC of 0.81. Among 112 participants with available urine specimens (34 of whom died), LA was the only urinary acid of significant independent prognostic value. The AUROCC for a model based on urinary LA alone was 0.72.Logistic regression models were developed for patients with severe malaria, with in-hospital death as the dependent variable, to assess the prognostic value of plasma and urine organic acid concentrations Table . An alteMetabolic acidosis with an increased SIG and high SBD is a prominent feature of severe falciparum malaria, and severity of the acidosis is closely linked to mortality , 16, 23.l-lactate isomers, whereas LC-MS detects both l- and d-isomers. Human metabolic pathways produce only l-lactate, but both isomers are produced by P. falciparum, as the parasite relies heavily on incomplete oxidation of glucose into LA for its own energy supplies [d-lactate can accumulate in the context of multiorgan failure, with increased transposition of bacteria from the ischaemic gastrointestinal tract [d-lactate can cause an encephalopathy in humans ranging from disorientation to coma [d-lactate could contribute to the pathogenesis of severe malaria requires further quantification and study.LA was detectable in plasma at a considerably higher concentrations than any other organic acid and was a strong independent predictor of death in patients with severe malaria, with a higher AUROCC than reported previously . We conssupplies . Bacterial tract –28, and  to coma . The hypl-tyrosine catabolism [Enterococcus faecalis, Escherichia coli, bifidobacteria and lactobacilli) can be a source of HPLA [HPLA was identified at higher concentrations in the plasma and urine of participants with severe malaria than in reference groups, and it was a strong and independent predictor of mortality in patients with severe malaria. Although total HPLA concentrations were relatively low, the high prognostic value of HPLA may reflect perturbations in associated metabolic pathways, with related acids also contributing to the total acid load. Alternatively, this may indicate strong biological activity of the anion at low concentrations or local accumulation in affected tissues. HPLA is a product of tabolism . Plasma tabolism , 31–35. tabolism , 36. Uritabolism . In thestabolism , 39. In  of HPLA . This co of HPLA –43. A poβ-HBA was high in plasma and urine of patients with severe malaria but highest in patients with severe sepsis. Concentrations were not independently correlated with mortality. β-HBA is synthesised in the liver from free fatty acids, and increased production of ketoacids can be related to the augmented metabolic demand, coupled with reduced oral caloric intake, in severe illness , 44, 45.Concentrations of α-HBA were high in patients with severe malaria, sepsis or encephalopathy, but they were not an independent predictor of mortality. α-HBA is a product of threonine and methionine catabolism, but it is also produced in glutathione synthesis during the conversion of cystathionine to cysteine. The glutathione pathway is upregulated to counteract oxidative stress, which is increased during severe malaria , 46. RaiWe acknowledge the limitations of this hypothesis-generating study and the need for further, targeted exploration of the mechanisms by which the identified acids influence the pathogenesis of malaria. The heterogeneity of organ dysfunction in both severe malaria and severe sepsis complicates our understanding of both conditions, particularly with regard to the metabolic pathways implicated. This heterogeneity reduces the statistical power of our analysis; yet, significant conclusions emerge.Concentrations of HPLA were significantly higher in patients with severe malaria than in patients with sepsis, febrile encephalopathy or uncomplicated malaria, which suggests a disease-specific role. HPLA also has prognostic significance independent of LA. Further research into the kinetics and biological effects of these acids, as well as into the contributing metabolic pathways, is warranted.Previously unmeasured organic acids could be identified in patients with severe falciparum malaria using a novel targeted LC-MS approach.A tyrosine metabolite and two ketoacids were detected at increased concentrations in patients with severe malaria.Lactate and 4-hydroxyphenyllactic acid were predictive of mortality in patients with severe malaria.Our findings highlight metabolic pathways potentially implicated in disease pathogenesis.


Plasmodium falciparum malaria in two sentinel sites in Senegal.In Senegal, a significant decrease of malaria transmission intensity has been noted the last years. Parasitaemia has become lower and, therefore, more difficult to detect by microscopy. In the context of submicroscopic parasitaemia, it has become relevant to rely on relevant malaria surveillance tools to better document malaria epidemiology in such settings. Serological markers have been proposed as an essential tool for malaria surveillance. This study aimed to evaluate the sero-epidemiological situation of P. falciparum infection was diagnosed using microscopy. P. falciparum antibodies against circumsporozoite protein (CSP), apical membrane protein (AMA1) and merozoite surface protein 1_42 (MSP1_42) were measured by ELISA method. A stepwise logistic regression analysis was done to assess factors associated with P. falciparum antibodies carriage.Cross-sectional surveys were carried out in Velingara  and Keur Soce  between September and October 2010. Children under 10 years old, living in these areas, were enrolled using two-level, random sampling methods. 42 and anti-CSP antibody was 38.12, 41.55 and 40.38%, respectively. The seroprevalence was more important in Velingara and increased with age, active malaria infection and area of residence.A total of 1,865 children under 10 years old were enrolled. The overall falciparum malaria prevalence was 4.99% with high prevalence in Velingara of 10.03% compared to Keur Soce of 0.3%. Symptomatic malaria cases (fever associated with parasitaemia) represented 17.37%. Seroprevalence of anti-AMA1, anti-MSP1_The use of serological markers can contribute to improved malaria surveillance in areas with declining malaria transmission. This study provided useful baseline information about the sero-epidemiological situation of malaria in Senegal and can contribute to the identification of malaria hot spots in order to concentrate intervention efforts.http://www.pactr.org).Trial registration number: PACTR201305000551876 ( Plasmodium falciparum antigens have been studied to assess malaria transmission and impact on the host immunity. To assess the level of malaria transmission, a pre-erythrocytic-stage antigen most commonly used is the circumsporozoite protein (CSP) with a short estimated half-life. Antibodies against this protein are correlated to transmission intensity and exposure duration, but not necessarily to plasmodial infections. This protein is labile and disappears quickly in the absence of exposure. P. falciparum erythrocytic-stage antigens, such as merozoite surface protein 1 (MSP) and apical membrane antigen (AMA1) with long half-lives, reflect the cumulative exposure to malaria and can be used as an indicator of the burden of malaria . Malaria prevalence was higher in Velingara at 10.03% [95% CI (8.3–12.7)] (90/866) than in Keur Soce, where it was 0.3% [95% CI (0.06–0.8)] (3/999). Among children with falciparum malaria, 17.37% had fever providing an odds ratio at 1.81 [95% CI (1.13–2.91)]  while in Keur Soce it was 72.07%. The proportion of subjects with fever (axillary temperature ≥37.5°C) at the time of survey was 20.4 and 14.71% in Velingara and Keur Soce, respectively. Overall 42 and CSP antibodies was 15.17 AU and 29.25 UA, respectively; both were higher in Velingara’s population (p < 10−3). Antibodies production was significantly higher among children with P. falciparum infection compared to non-infected children (Table The mean production of anti-AMA1 antibody was 16.04 AU and varied from 16.59 AU in Velingara to 15.57 AU in Keur Soce. No significance difference was noted between the two sites (p = 0.57). MSP1_n p < 10−. Antibod42 and anti-CSP antibodies was at 38.12% [95% CI (35.37–41.03)], 41.55% [95% CI (38.68–44.58)] and 40.38% [95% CI (37.54–43.36)], respectively. The seroprevalence of these antibodies was higher in Velingara compared to Keur Soce (p < 10−3) ; p value = 0.56] and in subjects living in Velingara 46.3% . The seroprevalence of anti-MSP1_42 antibody was 37.3%  in children under 5 years old while it was more important in children over 5 years old 47.57% .Multivariate logistic regression analyses revealed that seroprevalence increased with age, active malaria infection and area of residence. In children over 5 years old, the seroprevalence of anti-AMA1 antibody was 46.1%  and this was more important among female children (38.7%), subjects with stunting (37.97%) and subjects without anaemia (43.88%). Seroprevalence of anti-AMA1 antibody was higher in subjects with 42 antibody was 52.69% . For children over 5 years, the seroprevalence of anti-CSP antibody was 43.53%  compared to other children. Anti-CSP antibody was more important in female children (41%), children with stunting 43.72%  and children with anaemia (40.44%). The prevalence of anti-CSP antibody was associated with active malaria infection. Prevalence of anti-CSP antibody was more important in children with malaria infection 46.24% . The prevalence of anti-CSP antibody was more important in Velingara 52.66%  compared to Keur Soce (29.73%). There is no correlation between seroprevalence of antibody and sex . However, anaemia remained high in both sites. These data are consistent with results from national malaria indicator surveys conducted 2008–2009, which showed similar patterns in terms of P. falciparum carriage and anaemia prevalence [P. falciparum at 2.8% with disparities between the southern part (9.3%) and the central part (2.2%) of the country [This study was conducted to assess the sero-epidemiological situation of malaria in two sentinel sites with different epidemiological profiles in Senegal. The study showed a low prevalence of malaria parasitaemia, although  country . The dif country .P. falciparum, anaemia was closely associated with malaria parasitaemia. Other studies demonstrated that the main factors influencing anaemia occurrence in the central and the southern parts of the country are mainly represented by P. falciparum carriage, malnutrition, sickle cell traits and alpha-thalassaemia [Similar results were found in 2005 with the heterogeneity of malaria in the east and west of Cambodia . Despiteassaemia .42 and CSP antibodies was 38.12, 41.55 and 40.38%, respectively. Significant difference between the two areas was observed with a higher seroprevalence in the southern part (Velingara). Although the serological assessment confirmed malaria heterogeneity as shown by microscopy. Proportion of P. falciparum carriage was significantly lower than antibodies level. These findings are in accordance with what were observed in Madagascar [The overall seroprevalence of AMA1, MSP1_dagascar . In Tanzdagascar . Similardagascar . A high dagascar .These results show that serology could be a good indicator for malaria surveillance. 5% of the total study participant was found positive by microscopy while antibodies excretion increased by at least sixfolds. The study demonstrated that using microscopy alone for malaria surveillance could underestimate malaria burden, particularly in areas with reduced malaria transmission. These data are confirmed by other studies , 23, 27.42 and CSP antibodies increased with age, P. falciparum carriage and the area of residence. Similar results were found in Vanuatu in 2008 and in northern Peru between 2008 and 2010 [P. falciparum infection, the seroprevalence of antibodies is higher compared to those without P. falciparum infection. The association between malaria and level of antibody  has been demonstrated by several immuno-epidemiological studies [The study showed that seroprevalence of AMA1, MSP1_and 2010 , 29. In and 2010 . This waand 2010 , 31. In  studies –39. Comp studies .Gender, Hb level and nutritional status did not play a role in antibody production. This was demonstrated in 2002 in Senegalese preschool children when assessing the immunological consequences of intermittent preventive treatment .Serological markers can be a useful tool for malaria epidemiology characterization particularly in areas with a decrease of malaria and can even contribute to the identification of malaria hot spots in order to concentrate intervention efforts. Others studies suggest that sero-epidemiological analysis will be useful tool in assessing short-term changes in exposure and malaria transmission in area with a low or seasonal transmission. It was demonstrated in Ghana, Kenya and Indonesia , 41.The age of the study population being limited to 10 years constituted a study limitation. To better document the changing profile of malaria epidemiology, it would be relevant to extend the study to the other age groups in order to characterize the burden of the disease in the study areas.Serological markers can be used as complementary tools for malaria survey in areas with a declining pattern of malaria in Senegal. This study provided useful baseline information about the sero-epidemiological situation of malaria in Senegal and can contribute to the identification of malaria hot spots in order to concentrate intervention efforts.


P < 0.001) and 11% (P = 0.001) at every workload, respectively, accompanied by a 16% reduction in lactate concentration in plasma (P < 0.001), although all remained unchanged in the CNT trial (P > 0.2). All of the reductions were significantly greater in the ALA+SFC than the CNT trial (P < 0.05). Furthermore, the training days, impulse, and time at fast walking were 42% (P = 0.028), 102% (P = 0.027), and 69% (P = 0.039) higher during the ALA+SFC than the CNT intake period, respectively. Thus ALA+SFC supplementation augmented exercise efficiency and thereby improved IWT achievement in older women.A reduction in exercise efficiency with aging limits daily living activities. We examined whether 5-aminolevulinic acid (ALA) with sodium ferrous citrate (SFC) increased exercise efficiency and voluntary achievement of interval walking training (IWT) in older women. Ten women [65 ± 3(SD) yr] who had performed IWT for >12 mo and were currently performing IWT participated in this study. The study was conducted in a placebo-controlled, double-blind crossover design. All subjects underwent two trials for 7 days each in which they performed IWT with ALA+SFC  or placebo supplement intake (CNT), intermittently with a 2-wk washout period. Before and after each trial, subjects underwent a graded cycling test at 27.0°C atmospheric temperature and 50% relative humidity, and oxygen consumption rate, carbon dioxide production rate, and lactate concentration in plasma were measured. Furthermore, for the first 6 days of each trial, exercise intensity for IWT was measured by accelerometry. We found that, in the ALA+SFC trial, oxygen consumption rate and carbon dioxide production rate during graded cycling decreased by 12% ( ALA is an amino acid that can be found in many foods and is the sole initial material of heme biosynthesis in vivo , 37, 39.in vitro . However2 utilization efficiency in muscle mitochondria, it would increase the exercise efficiency and thereby improve the training achievement in older human subjects. To examine this hypothesis, we conducted a randomized, placebo-controlled, double-blind crossover study in older women.Based on these facts, we hypothesized that, if the oral ingestion of ALA accelerated the OThis study was approved by the Review Board on Human Experiments, Shinshu University School of Medicine, and conformed to the standards set by the Declaration of Helsinki. To minimize the effects of subject selection and the levels of training readiness, the subjects were recruited from the 547 participants in the “Jukunen Taiikudaigaku Project,” which is a health promotion program for middle-aged and older people in Matsumoto City, Japan. The participants had performed the interval walking training (IWT) program for more than 12 mo before this study; therefore, they were familiar with the exercise testing procedures used in the present study. Moreover, their exercise efficiency had likely reached the steady state and would thus enable us to detect only the effects of ALA intake on their exercise efficiency without accounting for the acute effects of exercise training.o2 peak). All subjects were nonsmokers and had no overt history of hepatic, thyroid, renal, metabolic, cardiovascular, or pulmonary disease, or orthopedic limitations in the exercise tests. Moreover, no subjects showed symptoms of iron deficiency anemia, as confirmed in the health examination before participating in the study. In the examination, the hemoglobin concentration ([Hb]), mean corpuscular volume, and mean corpuscular [Hb] were 14.1 ± 0.7 g/dl (mean ± SD), 90.5 ± 2.2 fl, and 33.8 ± 0.7 g/dl, respectively. Their physical characteristics were similar to those observed in our laboratory's previous study  gave written, informed consent before participating in this study. Each subject gave a complete medical history and underwent examinations of physical characteristics, resting blood pressure (BP), and peak aerobic capacity (V̇us study  and are days 1–7). Before and after the supplement intake period (days 0 and 8), subjects underwent a graded cycling test, during which the cardiorespiratory responses and lactate concentration in plasma ([Lac−]p) were measured. During the supplement intake period (days 1–6), except for the day before the graded cycling test (day 7), the training days, intensity, and time were recorded with a triaxial accelerometer   , 34, 52.o2) was increased. Therefore, we thought that a higher dose would be needed to improve mitochondrial function, if it occurred. Regarding dose safety, there is no legal dose limit in Japan because ALA phosphate has been approved as a food ingredient by the Pharmaceutical and Food Safety Bureau, the Ministry of Health, Labour and Welfare. Additionally, because the present study had a placebo-controlled crossover design, we could not test several doses of ALA+SFC.The composition of supplements  is shown in SFC, as a source of the iron ion, was contained in the supplements to enhance the final step of heme biosynthesis by ABCB6 transporter and ferrochelatase in mitochondria and to prevent the accumulation of heme biosynthesis intermediates, such as protoporphyrin IX, which might cause photodamage to the skin during outdoor exercise . AlthougP > 0.2). Moreover, the values generally met the recommended dietary allowance (RDA) for Japanese active older women . The results are shown in er women , and theer women , was verer women .P > 0.4). Subjects were asked to eat the standardized breakfast and lunch at normal time and to finish the standardized dinner by 2100. Additionally, subjects were asked to refrain from ingesting alcohol and caffeine during the day.On the day before the graded cycling test, which occurred four times : the total calories was 1,816 ± 16 kcal, the total protein was 66.6 ± 0.3 g, the total fat was 33.3 ± 0.1 g, and the total carbohydrate was 307 ± 4 g, with no significant differences observed before and after the supplement intake periods or between the CNT and ALA+SFC trials  atmospheric temperature and 50 ± 1% relative humidity. An 18-gauge Teflon catheter was then placed in the left antecubital vein for blood sampling. The catheter was connected to a 20-ml syringe that contained heparinized saline solution through an extension tube 2.0 ml in volume with a three-way stopcock.On the day of testing, subjects reported to the laboratory at 0900; they were normally hydrated but had not eaten any food for more than 12 h before the experiment. To ensure that they were well hydrated, they were instructed to drink 500 ml of tap water 2 h before the visit. Furthermore, they were instructed to ingest a supplement 2 h before the visit on e) every 15 s  to determine the V̇o2 and carbon dioxide production rate (V̇co2)  , Table 4o2 peak were a respiratory exchange ratio of >1.1, V̇o2 leveling off, despite increasing exercise intensity, and HR reaching the age-predicted maximal value. V̇o2 peak was determined by averaging the three largest consecutive values at the end of exercise. We also determined the gas exchange threshold during graded cycling according to the standard method   . Additioo2 and respiratory exchange ratio values during the last minute of each workload were averaged and used to calculate energy expenditure at a given workload. Under these conditions, efficiency was calculated based on the steady-state assumption that energy requirements are met by V̇o2 (1) V̇o2 during the last minute of each workload was relatively constant; and 2) when the values were plotted against workload, there was a linear relationship between them in each subject of each condition . Efficiency was then determined  represents V̇o2 at any time (t); V̇o2 BSLN is the baseline V̇o2 calculated as an average over 5 min before the onset of exercise; ΔV̇o2 is the steady-state increase in V̇o2 above the baseline V̇o2; τ represents the time constant defined as the time required to attain 63% of the ΔV̇o2; and TD represents the mathematically determined time delay for V̇o2 to start to increase after the onset of exercise. Briefly, after abandoning the phase I response of V̇o2 for the first 15 s to exclude the cardiodynamic effect , subjects were instructed to perform IWT, repeating five or more sets of 3 min of low-intensity walking at 40% of V̇o2 peak, followed by 3 min of high-intensity walking above 70% of V̇o2 peak per day for 4 or more days/wk, where training days, intensity, and time were recorded with a portable triaxial accelerometer (JD Mate) and transferred to the computer server at Shinshu University Graduate School of Medicine over the internet , which suggests no effects of weather conditions on the training achievement.Regarding the weather conditions on the first 6 of 7 days of supplement intake in the CNT and ALA+SFC trials, the precipitation from 0500 to 2000 was 1.7 ± 0.7 and 2.6 ± 0.8 mm/day, atmospheric temperature was 15.7 ± 1.1 and 13.9 ± 1.0°C, and relative humidity was 71 ± 1 and 75 ± 1%, respectively, with no significant differences observed between trials , period [the effect of stimulation order was present in CNT-(ALA+SFC) sequence group vs. (ALA+SFC)-CNT sequence group], and supplement effects. For the analysis, data were averaged during the graded cycling test, and changes after supplement intake (after-before) were calculated for each period. The three effects were then determined by the method reported by Chow and Liu . The nulP > 0.1). [Hb] before and after supplement intake was 13.3 ± 0.3 and 13.1 ± 0.3 g/dl in the CNT trial, respectively, and 13.1 ± 0.4 and 12.9 ± 0.4 g/dl in the ALA+SFC trial, respectively, with no significant differences between them . Additionally, V̇o2 peak and peak HR during the graded cycling test remained unchanged after supplement intake in both trials ; however, the peak workload tended to increase (P = 0.081). Furthermore, the time to exhaustion significantly increased by 0.8 min in the ALA+SFC trial (P = 0.022), although not in the CNT trial (P = 0.47) (P = 0.040).As shown in  = 0.47) ; the inco2, V̇co2, and V̇e responses during the graded cycling test. When comparing the responses obtained before and after supplement intake, an increase in V̇o2 after supplement intake was significantly attenuated by 12% for every workload 1 min after the start of exercise in the ALA+SFC trial (P < 0.001), although this did not occur in the CNT trial (P = 0.49). Similarly, an increase in V̇co2 and V̇e was significantly attenuated by 11% for every workload 2 min after the start of exercise in the ALA+SFC trial , although this did not occur in the CNT trial . We confirmed that the reduction in V̇o2 and V̇co2 after supplement intake was significantly greater in the ALA+SFC than in the CNT trial . In addition, there were no significant differences in HR and BP during the graded cycling test before and after supplement intake in either trial .NT trial , but the−]p during the graded cycling test. After supplement intake, the increase in [Lac−]p was significantly attenuated by 16% every workload in the ALA+SFC trial (P < 0.001), although this did not occur in the CNT trial (P = 0.59). We confirmed that the reduction in [Lac−]p after supplement intake was significantly greater in the ALA+SFC than in the CNT trial  vs. the (ALA+SFC)-CNT sequence group (n = 5)  was 0.93 ± 0.32 ml·kg−1·min−1. For the (ALA+SFC)-CNT sequence, the change in V̇o2 after supplement intake was −1.06 ± 0.26 ml·kg−1·min−1 in period 1 and −0.32 ± 0.45 ml·kg−1·min−1 in period 2, and the difference over time (periods 1−2) was −0.74 ± 0.56 ml·kg−1·min−1, which was significantly different from that observed in the CNT-(ALA+SFC) sequence (P = 0.032), thus indicating a significant supplement effect. Similarly, a significant supplement effect was observed in V̇co2 (P = 0.048) and [Lac−]p (P = 0.039), but was not observed in V̇e (P = 0.13). No carryover or period effects were observed in V̇o2, V̇co2, V̇e, or [Lac−]p (P = 0.52–0.97).The changes in respiratory responses to the graded cycling test were also examined by comparing the CNT-(ALA+SFC) sequence group ( (n = 5) , 26. ForP = 0.0086 for gross efficiency, P = 0.039 for net efficiency), although this did not occur in the CNT trial . On the other hand, we found no significant differences in delta efficiency before vs. after supplement intake in the ALA+SFC trial (P = 0.61).o2 responses to exercise, we determined the V̇o2 kinetics parameters at the onset of exercise. As shown in τ after supplement intake was significantly shortened (P = 0.026), and that ΔV̇o2 was significantly decreased (P = 0.0018) in the ALA+SFC trial, although this did not occur in the CNT trial , whereas TD remained unchanged after supplement intake in both trials .To assess the effect of ALA on transient V̇A), training impulse (B), and training time (C) during the supplement intake period (days 1–6). Training days were 42% higher in the ALA+SFC than the CNT trial (P = 0.028). The impulses at total and fast walking were 72% and 102% higher in the ALA+SFC than the CNT trial . The training time at fast walking was 69% higher in the ALA+SFC than the CNT trial (P = 0.039).A), training impulse (B), and training time (C) on each day in the period of supplement intake. The ratio in the ALA+SFC trial tended to increase after the 5th and 6th days, but with no significant differences from that in the CNT trial . Training impulse at fast walking in the ALA+SFC trial increased after the 3rd day, with significant differences from that in the CNT trial (P = 0.028). Similarly, training time at fast walking in the ALA+SFC trial increased after the 3rd day, with significant differences from that in the CNT trial (P = 0.039).o2, V̇co2, and [Lac−]p during the graded cycling test were significantly reduced at every workload with ALA+SFC supplementation, and that all of the reductions were significantly greater in the ALA+SFC trial than in the CNT trial. In addition, training days, training impulse, and training time at fast walking were significantly higher in the ALA+SFC trial than in the CNT trial.Although ALA has been reported to enhance the production of heme and cytochromes in vitro , 39 to ao2 during the graded cycling test was significantly reduced by 12% at every workload in the ALA+SFC trial, but not in the CNT trial, with 12% increases in gross and net efficiencies with significance. Regarding the mechanisms, Ferguson et al.  with age and suggested that the phosphorylative-coupling efficiency (ATP/V̇o2) rather than contractile-coupling efficiency (work/ATP) played a key role in the reduced cycling efficiency with age. In the present study, we first confirmed in the whole body of older human subjects that the oral ingestion of ALA increased the gross and net efficiencies during cycling by 12% at the cadence mentioned above; however, we found no significant increase in delta efficiency, reflecting in part the phosphorylative-coupling efficiency, in the ALA+SFC trial  and ΔV̇o2 is an indicator of an O2 deficit  has been recommended in recent guidelines to increase V̇o2 peak in older people .This study was supported by a grant from the No conflicts of interest, financial or otherwise, are declared by the author(s).Author contributions: S.M., A.M., Y.-i.K., S.I., K.T., T.T., M.N., and H.N. conception and design of research; S.M., A.M., Y.-i.K., S.I., Y.K., Y.O., E.S., and H.N. performed experiments; S.M., A.M., and S.I. analyzed data; S.M., A.M., Y.-i.K., S.I., and H.N. interpreted results of experiments; S.M. and A.M. prepared figures; S.M., A.M., and H.N. drafted manuscript; S.M., M.N., and H.N. edited and revised manuscript; S.M., A.M., Y.-i.K., S.I., Y.K., Y.O., E.S., K.T., T.T., M.N., and H.N. approved final version of manuscript.


Plasmodium infection in blood donors from a blood transfusion centre in São Paulo, Brazil.In Brazil, malaria is endemic in the Amazon River basin and non-endemic in the extra-Amazon region, which includes areas of São Paulo state. In this state, a number of autochthonous cases of malaria occur annually, and the prevalence of subclinical infection is unknown. Asymptomatic infections may remain undetected, maintaining transmission of the pathogen, including by blood transfusion. In these report it has been described subclinical Plasmodium falciparum and Plasmodium vivax. Subclinical malaria cases were geo-referenced.In this cross-sectional study, representative samples of blood were obtained from 1,108 healthy blood donors at the Fundação Pró-Sangue Hemocentro de São Paulo, the main blood transfusion centre in São Paulo. Malaria exposure was defined by the home region . Real-time PCR was used to detect Plasmodium; 57 of these were infected by P. falciparum, 25 by P. vivax, and 2 by both. The prevalence of P. falciparum and P. vivax was 5.14 and 2.26, respectively. The overall prevalence ratio (PR) was 3.23  2.03, 5.13); P. falciparum PR was 16.11  and P. vivax PR was 0.47 . Plasmodium falciparum subclinical malaria infection in the Atlantic Forest domain was present in the mountain regions while P. vivax infection was observed in cities from forest-surrounded areas.Eighty-four (7.41%) blood donors tested positive for Plasmodium in healthy blood donors from a region known as non-endemic, which is important in the context of transfusion biosafety, was described. Infected recipients may become asymptomatic carriers and a reservoir for parasites, maintaining their transmission. Furthermore, P. falciparum PR was positively associated with the forest environment, and P. vivax was associated with forest fragmentation.The presence of  Plasmodium vivax, which accounts for more than 80% of identified cases; Plasmodium falciparum is responsible for 16.3%, and Plasmodium malariae is associated with a small number of cases [Anopheles darlingi is the primary vector in recently deforested areas [In Brazil, human malaria is mainly caused by of cases . The majed areas .Anopheles mosquito bites, in a specific time frame. In endemic regions, this index is spatially and temporarily variable, and its values can be low (0.1–9.9), moderate (10.0–49.9), or high (>50) [Plasmodium spp; however, most of them showed low levels of parasitaemia [The annual parasite index is typically employed to estimate the risk of occurrence of malaria in human populations that are exposed to infective gh >50) , dependi0 , depengh (>50) . In the gh (>50)  and in rgh (>50)  in the s low 0.1–.9, modersitaemia ,10,11. Tsitaemia .Plasmodium pathogens that cause malaria in humans are naturally transmitted by infectious Anopheles mosquito bites [P. vivax[Plasmodium infection in blood donors from an endemic region are distinct from those adopted in non-endemic areas [Plasmodium infection [Plasmodium spp. in non-endemic areas and the fact that there is no obligation for screening for Plasmodium, it is plausible to suppose that accidental transfusional Plasmodium transmission can occur, mainly by the presence of subclinical infection in humans.to bites . Howeverto bites  and intrto bites . The firto bites -17. Althto bites ,19. In t[P. vivax when theic areas . Furthernfection . ConsidePlasmodium parasites represent an important risk factor because they can cause severe malaria with a high fatality rate [P. falciparum, up to seven years for P. vivax, and even for an entire lifetime for P. malariae[. Consequently, an asymptomatic human who is unaware of his/her status as a Plasmodium reservoir can transmit the parasites when donating blood. Usually, parasite concentration is low in asymptomatic individuals [Anopheles females [Transfusion-transmitted ity rate . The chr malariae. Consequividuals ; however females  and caus females .P. falciparum and P. vivax among blood donors at a public blood bank in the municipality of São Paulo, Brazil; 2) the spatial distribution of the blood donors; and 3) the potential association between the geographical origins of donors and the Plasmodium species.Employing a high sensitivity real-time PCR in a cross-sectional design, it was addressed 1) the prevalence of A cross-sectional study was adopted with the main objective of estimating the prevalence of subclinical malarial infections among blood donors in the State of São Paulo. The studied population was composed of 1,108 blood donors enrolled at the Fundação Pró-Sangue Hemocentro de São Paulo, Brazil, from August 2008 to March 2010. The sampling procedure employed a non-probabilistic cumulative method. Accordingly, individuals who came to donate blood were cumulatively selected regarding free potentially past/recent malarial infections as inclusion criteria. Each blood donor volunteer was interviewed for previous malaria, location of residence, and travel/visit to endemic regions. Brazilian guidelines recommend refusing as a potential blood donor any individual who has lived in a malaria-endemic region within the past three years as well as those who have travelled to those areas in the past six months. Consequently, the present study does not include individuals who were rejected for blood donation based on these conditions. In terms of sampling procedure, it was knew who would be next to donate blood nor who would be included in the study. This circumstance made the sampling procedure random, which is a reasonable proxy for obtaining a representative sample of blood donors from the population in the State of São Paulo .Plasmodium spp. prevalence (1.3%) in blood banks in the State of Amazon [To calculate the sample size, the prior prevalence of subclinical malarial infections among blood donors was considered to be 1%. This subclinical malaria information is unavailable for the State of São Paulo, and the sample size for the cross-sectional study thus was estimated using alence 1.% in bloon* = minimum sample size; z = critical percentile of the z distribution (1.96); α = significance level (0.05); ϵ = standard error or tolerable error (0.02 or 2%), which measures discrepancy between population prevalence (unknown) and estimated prevalence from the obtained sample; and P = prior prevalence data (as supported by [where orted by -26). BasAnopheles mosquito vectors). Individuals living in forested areas of the Atlantic Forest were considered at risk of infection whereas those from urban areas in the Metropolitan Region of São Paulo were considered to be without risk of malaria  was obtained, four groups were defined: disease-exposed (a), non-disease exposed (b), disease non-exposed (c), and non-disease non-exposed (d). Exposure status was defined based on evidence of risk of being in contact with infectious mosquito bites  − 1  was estimated, using the following equations:In this cross-sectional study, the starting point was a representative sample (N) obtained for defining the four groups under study . First, the prevalence of P. vivax, and pf = P. falciparum. N is the representative sample. The prevalences  represented estimates of the magnitude of the malarial subclinical infections on the blood donor population. Then the PR was estimated, giving a measure of the prevalence of subclinical malarial infections in the exposed group in relation to the non-exposed group, using the following equation:where a + c is the number of individuals carrying the malaria parasite, and t = all parasites, pv = P. vivax, and pf = P. falciparum. If the presence of a certain exposure factor  does not increase the prevalence of subclinical malaria, the PR is expected to be 1; PR > 1 indicates a positive association between exposure and prevalence; and a PR < 1 suggests that the association is negative. A confidence interval of 95% was provided for each estimate of the PR. Additional theoretical rationale for these procedures can be obtained from Rothman [where a = the number of subclinical malarial infections in the exposed group; c = the number of subclinical malarial infections in the non-exposed group; a + b = total exposed individuals; c + d = total non-exposed individuals. PR was estimated for each parasite, with t = all parasites, pv =  Rothman .Plasmodium infection cases were geo-referenced using resident address and routines implemented in the R 3.0.0 programming environment with the aid of the ggmap package [TM. These searches returned geographic coordinates (longitude and latitude) that were plotted over a satellite imagery scenario provided by Google MapsTM. Geo-referenced data were used to identify possible explanations considering the observed pattern of geographic distributions of subclinical malarial infections.The detected subclinical  package . With thA total of 1,108 individuals were selected in the present study. From this sample, 61 were exposed and had a subclinical malaria infection; 439 were exposed and did not have a subclinical malaria infection; 23 were non-exposed and had a subclinical malaria infection; and 585 were non-exposed and did not have a subclinical infection . Considering only P. falciparum subclinical infections, 53 blood donors of the exposed group were positive for subclinical infection, 447 were exposed and did not have any evidence of infection, 4 individuals of the non-exposed group tested positive, and 604 tested negative for infection ; the prevalence among exposed individuals was 10.60 (%); and among non-exposed, it was 0.66 (%)  , 57 were Plasmodium vivax subclinical infections, seven exposed participants had a subclinical infection, 493 were exposed without subclinical infection, 18 non-exposed had subclinical infection, and 590 non-exposed did not have subclinical infection (Table P. vivax infection among blood donors was 2.26 (%); prevalence among exposed individuals was 1.40 (%), and among non-exposed, it was 2.96 (%)   were from the mountains . In both municipalities, forest cover is greater than 50%, and landscape fragmentation provides high levels of interaction between the forest and the urban, anthropogenic environment  and São Lourenço da Serra  are primarily rural locations embedded in a well-preserved and extensively forested area on the slope of Serra do Mar, where the vectors are mainly sylvatic mosquitoes from the subgenus Kerteszia of Anopheles. The human population from these areas was here defined as the exposed population whereas inhabitants of urban areas intercalated by forest fragments formed the non-exposed population. In the non-exposed population, the PR of P. vivax revealed a positive  between the parasite infection and urban inhabitants. However, this result may arise from the fact that the residences of the blood donors are situated in urban areas at the edge of forest fragments. Consequently, the presence of P. vivax is likely to be associated with forest fragments and involves both Kerteszia and Nyssorhynchus species, or even other Anopheles subgenera [Plasmodium species that cause malaria in humans is a dynamic process that involves ecological, environmental, and climate factors as well as social, political, and economic determinants. The high prevalence of Plasmodium spp. subclinical infections in blood donors living either in forested areas or urban regions intermixed with forest fragments suggests that some evolutionary mechanisms are modulating the virulence of P. falciparum and P. vivax in a way that does not negatively affect survival of the parasite, humans, mosquitoes, and non-human primates that seem to be involved in malaria transmission in the Atlantic Forest. The dynamics of human malaria transmission in the Brazilian Amazon forest seems to involve similar evolutionary mechanisms that lead to subclinical infections in humans [An. darlingi) [P. falciparum[Alouatta monkeys have been found to be infected by P. vivax and P. malariae[P. falciparum[annually . Becauseservoirs ,49, malaubgenera ,50. The n humans , and traarlingi) , and nonalciparum. In the alciparum.The scientific novelty and translational importance of the present results are two-fold: 1) They could represent a serious problem for the blood transfusion programme in the State of São Paulo; and 2) they could be evidence of evolutionary mechanisms of attenuation of malarial symptoms in humans.The translational importance of the first aspect is obvious. Because some infected recipients may become asymptomatic carriers and constitute a reservoir for parasites, maintaining their transmission, further investigations are needed to better document the risk of transfusion-transmitted malaria in these regions and to establish whether a specific malarial infection test should be considered for blood donors who live in or near the Atlantic Forest. The second aspect is more subtle and needs more data before being established as a paradigmatic cornerstone for the evolution of malaria transmission dynamics. In terms of clinical and economic viewpoints, the Atlantic Forest’s dynamics of malaria transmission could be a real-world example of symptomatic malaria elimination.P. falciparum and P. vivax infection in healthy blood donors from São Paulo a non-endemic region was described. This finding is a significant concern in the context of transfusion biosafety. Some infected recipients may be asymptomatic carriers, constituting a reservoir for parasites, maintaining their transmission and increasing the risk of transfusion-transmitted malaria in these regions. Additionally, prevalence ratio infection values showed a positive association between forest environment and P. falciparum infection, which was linked to residence in the mountain region of the Atlantic Forest domain. Moreover, P. vivax seemed to be associated with forest fragmentation because it was identified in residents of cities in forest-surrounded areas. These results indicate a link between the pathogen prevalence and the Atlantic Forest environment.The presence of subclinical The authors declared that they have no competing interests.AMM, PEDL and SPB conceived the idea. LMFM, DL, GZL, AMM, MAMS and SPB participated in the study design and drafted the manuscript. AMM and PDLC supervised sample collection. LMFM, DL, GZL, LAF, MFFC and CTDR performed the experiments. LMFM, DL, GZL, AMM, CTDR, PEDL, MAMS and SPB analysed and interpreted the data. All authors read and approved the final version of the manuscript.Primers and probes used for detection of P. falciparum and P. vivax by real-time PCR. Description: The data provided the primers and probes used to perform real-time PCR.Click here for file


Iron deficiency affects one quarter of the world's population and causes significant morbidity, including detrimental effects on immune function and cognitive development. Accordingly, the World Health Organization (WHO) recommends routine iron supplementation in children and adults in areas with a high prevalence of iron deficiency. However, a large body of clinical and epidemiological evidence has accumulated which clearly demonstrates that host iron deficiency is protective against falciparum malaria and that host iron supplementation may increase the risk of malaria. Although many effective antimalarial treatments and preventive measures are available, malaria remains a significant public health problem, in part because the mechanisms of malaria pathogenesis remain obscured by the complexity of the relationships that exist between parasite virulence factors, host susceptibility traits, and the immune responses that modulate disease. Here we review (i) the clinical and epidemiological data that describes the relationship between host iron status and malaria infection and (ii) the current understanding of the biological basis for these clinical and epidemiological observations. Iron deficiency and malaria are significant co-morbidities in large portions of the developing world, and both maladies disproportionately affect pregnant women and children. Malaria causes an estimated 250 million infections and 500,000 deaths annually. Iron deficiency is estimated to affect one quarter of the world's populations causing substantial morbidity. Fortunately, iron deficiency is easily treated with iron supplementation  have made the clinical and epidemiological studies difficult to interpret  is indicative of iron deficiency. However, ferritin is also an acute phase protein which is elevated in the context of infection, and as a result is not a reliable marker of human iron status in the presence of infection or inflammation. Like serum ferritin, transferrin saturation and transferrin receptor levels are biochemical markers of human iron status that are also sensitive to infection and inflammation. As a result evaluating an individual's iron status during an infection has proven difficult . During the erythrocytic stage of infection, the parasite repeatedly invades, replicates within, and egresses from host RBCs. This erythrocytic stage of infection is responsible for all symptoms of disease  the protection of iron deficiency against malaria and (ii) the increased risk of malaria associated with iron supplementation is critical for managing iron supplementation campaigns in malaria endemic regions.2+) and ferric (Fe3+), makes it indispensable for many critical biological processes, including DNA replication, cellular respiration, and oxygen transport. However, the same useful biphasic properties of iron which make it indispensable also contribute to its high cytotoxicity. As a result the human host tightly regulates iron availability and usage.Iron is an essential nutrient for nearly every living organism including humans and the malaria parasite. Iron impacts a broad range of biological processes; including host and parasite cellular function, erythropoiesis and immune function. The capacity of iron to fluctuate between two oxidation states, ferrous  can confer resistance to artemisinin. The authors speculate that these mutations cause a disruption of the parasite's ability to detoxify ROS because the efficacy of artemisinin depends on its ability to generate oxygen radicals and some kelch-containing proteins in other organisms have been shown to be involved in the regulation of cytoprotection  and their accompanying iron responsive elements are critical for maintaining cellular iron homeostasis in the human host. IRPs and iron responsive elements are responsible for mobilizing iron when demands are high and moving iron into storage when excess iron may promote ROS formation , in iron containing proteins (400 mg), in serum bound to transferrin (3–7 mg), and in storage proteins such as ferritin 1 g). Host iron reservoirs available to erythrocytic stage malaria parasite include: (1) transferrin and non-transferrin bound iron (NTBI) in the serum and (2) intra-erythrocytic iron contained within hemoglobin, ferritin, as well as trace amounts freely bioavailable iron in the RBC cytosol , NTBI is scarce in healthy individuals. There is strong evidence that transferrin associates with pRBCs but not uninfected RBCs. Work by Pollack et al. shows that pRBCs take up Fe59 bound to human transferrin, and a recent publication by our own group demonstrates that incubation of pRBCs with transferrin and ferric citrate increases the bioavailable iron in pRBCs  is especially intriguing Table . BecauseP. falciparum laboratory lines. It is possible laboratory lines have adapted to an iron-starved extracellular environment. Furthermore, because hemoglobin is an essential nutrient for erythrocytic stage malaria, it is impossible to “starve” the parasite of iron in vitro and this may in turn limit the ability to study the effect of serum iron on P. falciparum.Despite strong evidence that transferrin associates with pRBCs, neither iron depletion nor iron supplementation of malaria culture media has any observable effect on parasite growth  as well as iron stored within ferritin (0.7 nM), and it is possible that the parasite is capable of utilizing one or both of these erythrocyte iron reservoirs. Currently, however, there is no reported evidence to either support or refute these possibilities  the malaria parasite acquires remains unclear, all the potential host iron reservoirs (serum and intra-erythrocyte) available to erythrocytic stage malaria are affected by iron deficiency as well as iron supplementation. Therefore, it is reasonable to hypothesize that iron deprivation and excess iron contribute to the relationship between host iron and malaria risk observed in the clinical studies discussed earlier. That said, even during iron deficiency, the erythrocytic stage of the parasite inhabits the most iron rich environment in the human body. As such it is alternatively possible that neither iron deficiency nor iron supplementation perturb iron reservoirs enough to significantly impact the parasite.in vitro; it is reasonable to hypothesize that that elevated zinc protoporphoryin IX in iron deficient erythrocytes provides protection against malaria infection by impeding parasite growth . However, P. vivax is not the only Plasmodium species that prefers young RBCs. In fact many species of Plasmodium, including P. falciparum, preferentially infect young RBCs, and furthermore young RBC support greater parasite replication than more mature RBCs  iron deficiency and protection from malaria infection and (ii) iron supplementation and increased risk of malaria. However, there is still much to be learned. Furthermore, study of the competition between the malaria parasite and the human host for iron can serve as a translational model to identify critical molecular mechanisms of Martha A. Clark, Morgan M. Goheen, and Carla Cerami wrote and edited the manuscript. All authors have read and approved the final manuscript.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.


We screened 52 children adopted from Ethiopia for malaria because they had previously lived in a disease-endemic region or had past or current hepatomegaly or splenomegaly. Seven (13.5%) children had asymptomatic malaria parasitemia by microscopy (n = 2) or PCR (n = 5). Our findings suggest that adoptees at risk for asymptomatic malaria should be screened, preferably by PCR. Plasmodium falciparum, P. vivax, and, less frequently, P. ovale is endemic to several regions in Ethiopia ; intestinal parasites ; hepatitis B or C virus; HIV; and syphilis  during February 2006–June 2011 for results of standard infectious disease screening tests recommended by the American Academy of Pediatrics: tuberculosis , and none had thrombocytopenia. All children with a positive blood smear or PCR result were treated: atovaquone/proguanil for P. falciparum infections, chloroquine followed by primaquine for P. vivax infections, and atovaquone/proguanil followed by primaquine for the mixed infection and infections with no species identified.During the period studied, 255 international adoptees from Ethiopia were seen at the clinic. Adoptees’ mean age at medical evaluation was 2.8 years (range 3.4 months–14.9 years); 148 (58%) were female and 107 (41.9%) were male. All 255 children were asymptomatic for malaria, but 52 met malaria screening criteria and were tested by peripheral blood smear (n = 24), PCR (n = 24), or both (n = 4). Of the 52 children, 7 (13.5%) had blood smear (2 children) or PCR (5 children) results positive for Giardia intestinalis flagellates were most common , followed by Blastocystis hominis protozoa   had positive results; , 15.7%) . Evidenc, 15.7%) .G. intestinalis flagellates)  of 52 adoptees from Ethiopia who had lived in a malaria-endemic region or had hepatomegaly or splenomegaly by clinical history or on physical examination had asymptomatic malaria parasitemia. We also confirm the findings of previous studies that showed high rates of infection with intestinal parasites  or past or current splenomegaly. However, a limitation of our study is that we used essentially these criteria to screen, and the prevalence of asymptomatic malaria might have differed if we used different criteria. For example, we did not screen all children with anemia (hemoglobin level of <11 g/dL); because malaria is a leading cause of anemia in malaria-endemic areas, anemia may be a useful additional screening criterion for malaria.Plasmodium species that cause disease in humans and can provide species identification at low levels of parasitemia. In our study, microscopy testing on 2 children failed to determine the malaria species, a common difficulty in persons with low-level parasitemia. Without knowing the malaria species, we had to treat the children for both P. falciparum and P. vivax infection, which involved testing for glucose-6-phosphate dehydrogenase deficiency and treatment with multiple antimalarial medications. Knowledge of the prevalent species in immigrants from a specific area can also inform public health efforts and prophylaxis planning for travelers to that area. For these reasons, PCR is likely the test of choice for detection of asymptomatic parasitemia in children adopted from malaria-endemic areas.For over a century, microscopy has been the standard for documentation of malaria infection in persons with clinical malaria, but PCR has greater sensitivity for detection of low-level parasitemia  must have microscopy or rapid diagnostic testing performed immediately, because these results are typically available quickly and can guide immediate decisions regarding treatment.We now use PCR to screen asymptomatic children from malaria-endemic areas. We recommend this method for centers with rapid access to PCR for all 5 human P. falciparum and P. vivax parasitemia. The study findings support the importance of obtaining a careful history to determine malaria risk and conducting PCR screening for asymptomatic infection in children with the noted risk factors. These findings may also be relevant to children adopted from other malaria-endemic countries.In summary, this study shows that children adopted from Ethiopia who lived in malaria-endemic regions of Ethiopia or had past or current splenomegaly are at risk for asymptomatic


Many studies report associations between human genetic factors and immunity to malaria but few have been reliably replicated. These studies are usually country-specific, use small sample sizes and are not directly comparable due to differences in methodologies. This study brings together samples and data collected from multiple sites across Africa and Asia to use standardized methods to look for consistent genetic effects on anti-malarial antibody levels.4] plus total IgE were measured by ELISA techniques. Regression models were used to investigate the associations of clinical and genetic factors with antibody levels.Sera, DNA samples and clinical data were collected from 13,299 individuals from ten sites in Senegal, Mali, Burkina Faso, Sudan, Kenya, Tanzania, and Sri Lanka using standardized methods. DNA was extracted and typed for 202 Single Nucleotide Polymorphisms with known associations to malaria or antibody production, and antibody levels to four clinical grade malarial antigens [AMA1, MSP1, MSP2, and (NANP)4 epitope of the pre-erythrocytic circumsporozoite protein, while there was little or no correlation with total IgE levels. Individuals with sickle cell trait had significantly lower antibody levels to all blood-stage antigens, and recessive homozygotes for CD36 (rs321198) had significantly lower anti-malarial antibody levels to MSP2.Malaria infection increased levels of antibodies to malaria antigens and, as expected, stable predictors of anti-malarial antibody levels included age, seasonality, location, and ethnicity. Correlations between antibodies to blood-stage antigens AMA1, MSP1 and MSP2 were higher between themselves than with antibodies to the (NANP)Although the most significant finding with a consistent effect across sites was for sickle cell trait, its effect is likely to be via reducing a microscopically positive parasitaemia rather than directly on antibody levels. However, this study does demonstrate a framework for the feasibility of combining data from sites with heterogeneous malaria transmission levels across Africa and Asia with which to explore genetic effects on anti-malarial immunity.The online version of this article (doi:10.1186/s12936-015-0833-x) contains supplementary material, which is available to authorized users. Despite many years of research and intervention, malaria remains a major global health concern. Approximately half of the world’s population is at risk, and in 2013 there were 207 million estimated cases of malaria and 627,000 deaths )At the outset investigators selected a core set of clinical parameters to be collected by all sites, taking into account variations in study design and implementation, and agreed on methods for recording these clinical measurements to encourage uniformity across the data. These clinical parameters were chosen based on their previous association with malaria infection or anti-malarial immune responses  and assays with poor performance or concordance were removed from the multiplex during testing. A total of 202 SNP-assays were designed. These included a set of 65 that have been analysed in a severe malaria case–control study across 11 countries [DNA extraction was performed at each site using the local method of choice; either Nucleon™ BACC Genomic Extraction Kits  or Qiagountries  plus a fP. falciparum erythrocytic stage parasite proteins apical membrane antigen 1 (AMA1), merozoite surface protein 2 (MSP2) and merozoite surface protein 1, 19 kDa fragment (MSP119). In addition, antibodies to a synthetic peptide (NANP)4 representing the major B cell epitope repeat of the circumsporozoite protein (CSP) of P. falciparum, and the amount of total IgE, were measured. Assays were carried out using a single uniform combined protocol for all samples.All study sites supplied serum or plasma samples to a central repository established at the National Institute for Biological Standards and Control (NIBSC). Samples were divided into two aliquots and stored at −80 °C in individually barcoded tubes  and racks. Before storage, a 5-µl aliquot was removed and diluted to 50 µl in PBS-0.2 % (w/v) sodium azide in a storage plate in order to characterize each sample. This characterization took the form of assaying specific antibody levels to the recombinant 19 (Wellcome sequence) [19 of Shirley Longacre . The 16 residue synthetic peptide (Asn-Ala-Asn-Pro)4 (NANP)4 was a kind gift of Eric Tongren .Recombinant AMA1 (3D7 sequence) , MSP2 (3equence)  were allP. falciparum standard on each plate coated with malaria antigen and the IgE reference serum, 75/502 (NIBSC), was used for IgE determinations. The negative control serum was a pool of 40 European individuals who had never been exposed to malaria.A reference plasma pool obtained from 20 malaria-exposed adults in the Gambia (Brefet4 pool)  was used19 and (NANP)4) or anti-human IgE MAb  (50 µl of 1 µg/ml), incubated overnight at 4 °C, washed three-fold with PBS-0.05 % Tween 20 (PBS/T) , blocked with 200 µl of blocking solution (2 % skimmed milk powder in PBS/T) for 3 h at ambient temperature and washed three times with PBS/T. Samples of each characterization sample (see above) were diluted in blocking solution and aliquots added in duplicate to plates as follows : 50 µl of 1:200 final dilution for (NANP)4-coated plates; 50 µl of 1:1,000 final dilution for MSP119, MSP2 and IgE and 100 µl of 1:2,000 for AMA1 plates. After overnight incubation at 4 °C, plates were washed six times with PBS/T, 50 µl of horseradish peroxidise-conjugated rabbit anti-human IgG (DAKO)  added to each well and plates incubated for 3 h at room temperature. Following six-fold washing in PBS/T, 100 µl of Sigma-Fast o-phenylenediamine dihydrochloride (OPD) reagent solution (Sigma) was added to each well. Plates were developed at room temperature for 10–15 min (20–30 min for (NANP)4 ELISA), the reaction stopped by addition of 25 µl 2 M H2SO4 and plates read in a plate reader  at 492 nm. A standard curve was fitted to the reference serum data obtained for each antigen as previously outlined [19 and MSP2, using independently expressed preparations of the same antigens  that were not measured correctly due to technical difficulties. These were removed and excluded from further analyses. Plots of the residuals from the regression analyses (see below) were made , Burkina Faso and Tanzania studies collected data during their malaria transmission season, Mali (Pongonon) included only individuals positive for malaria, Senegal and Sudan based data collection on active case detection, Kenya collected data as part of a birth cohort study at aged 7 years-old, and Sri Lanka followed up individuals that had been malaria positive in 1992/93. Further details can be found in Table 2 range = 0.21–0.35), followed by correlations between these antibodies and anti-(NANP)4 (r2 range = 0.11–0.24), with total IgE showing little or no correlation with any of the anti-malarial antibodies  was not observed in Sri Lanka but was at a frequency >0.99 in the African populations. Differences in allele frequencies between East and West African populations were also observed: the CD36 gene SNP (rs3211938) had a mean derived allele frequency of 28.9 % (range 0.7–61.9 %) in West Africa compared to 7.2 % (range 2.7–9.8 %) in East Africa; the HBB gene SNPs  had mean derived allele frequencies of 3.5 % (range 1.5–4.6 %) and 5.5 % (range 0.7–10.2 %), respectively, in West Africa compared to 5.9 % (range 1.6–7.7 %) and 0.006 % (range 0.0–0.003 %) respectively in East Africa. SNPs that were polymorphic in East Africa but not in West Africa included three RAD50 gene SNPs , one IL4 SNP (rs4986964), one IFNGR1 gene SNP (rs11575936) and two STAT6 gene SNPs . An FCER2 gene SNP (rs35825847) was the only SNP polymorphic in West Africa but not in East Africa.Allele frequencies in Sri Lanka were generally different to those observed at African sites  was rs334 in HBB, encoding sickle haemoglobin. Compared to HbAA, HbAS individuals had lower concentrations of antibodies to merozoite antigens AMA1 , MSP1  and MSP2 . No association of HbAS was seen for anti-(NANP)4 antibodies.Figure CD36) had significantly lower concentrations of antibodies to MSP2 .A candidate SNP in CD36 came close to the Bonferroni-corrected threshold, which is probably over-conservative. Compared to ancestral homozygotes and heterozygotes combined, recessive homozygotes at rs3211938 (−4). rs156029 (P4HA2) showed heterozygous association with anti-AMA1 levels . rs542998 (RTN3) showed recessive association with anti-MSP1 levels . No associations were found for antibody levels to (NANP)4  showed a recessive association with anti-MSP1 levels . The results of meta-analysis for the 4 marginal genes  are shown in Additional file In order to look at the consistency of effect we have made forest plots of the betas. Figure Given the association between sickle and anti-malarial antibody levels (see above), a regression analyses was conducted between malaria infection (microscopy result and parasite density) and sickle cell trait. HbAS individuals were significantly protected from becoming microscopically positive for malaria infection  at the time of sampling as measured by microscopic positivity and two had an ascertainment design not allowing an analysis of effect of age on malaria prevalence . Across the remaining studies, there was a significant increase in the odds of having microscopically positive malaria from the 1 year-old to 5–15 years-old age groups , after which prevalence declined into adulthood . This is similar to that observed at individual site studies –31. ManyMicroscopic malaria prevalence at a site did not always predict the magnitude of the anti-malarial antibody response. In Sri Lanka falciparum and vivax prevalence has been decreasing to almost zero by the time of plasma collection for this study  yet anti2 > 0.3), and showed less correlation with anti-(NANP)4 antibodies (r2 between 0.2 and 0.3) which are reflective of exposure to pre-erythrocytic parasites (sporozoites and liver stages). All anti-malarial antibodies showed similar distributions and peak titres from 1:100 to 1:1,000. Total IgE levels were not correlated with any of the other antibody levels. Although there was a marginally significant increase in total IgE levels with malaria infection, it was not possible to corroborate whether this was due to polyclonal induction of IgE antibodies during malaria infection [As expected, antibody titres to asexual blood stage antigens were most highly correlated with each other  with reduced antibody titres to merozoite antigens. Previous research has been inconsistent regarding the relationship between sickle cell trait and IgG responses to malaria: some studies have reported higher antibody levels in HbAS compared to HbAA individuals , whilst a et al.  concludeThe most likely mechanism for lowered antibody levels in HbAS individuals is by reducing the net exposure of the immune system to parasites; for example, by reducing invasion and development of parasites in HbAS erythrocytes , 54, enhIndeed across 21 published studies, the majority having less than 1,000 individuals, there is no consensus on the effect of HbAS on parasite density or prevalence, although the weight of evidence is probably for no effect . It is There are several features of this study that increase the reliability of the associations found between HbAS and merozoite antibodies. Firstly, a large dataset was generated with over 13,000 individuals, 12,380 of whom were HbAA and 868 HbAS. Secondly, a similar effect size was found for HbS in association with anti-AMA1, anti-MSP1 and anti-MSP2 levels, using the heterozygote model. Thirdly, the p-values for each HbAS-antibody association were similar and highly significant even after adjusting for multiple testing. Finally, not including an interaction term between SNP and study in the model meant that all significant outcomes represented effects that were consistent across sites, as demonstrated in the Forest plots for HbAS with the merozoite antibodies. The only exceptions were the Manteourou study in Mali and the Sri Lanka study, which had opposite directions of effect to the other sites for anti-AMA1. Neither of these associations were significant and were likely due to relatively small sample sizes  combined with low derived allele frequencies for HbAS .−4.One SNP was found to be marginally associated with antibody levels to merozoite antigens. The SNP in CD36 (rs3211938) on chromosome 7 was associated with a reduction in anti-MSP2 levels using the recessive genetic model, which is consistent with the reduced anti-MSP1 levels observed in individuals recessive homozygous for this SNP in another area of Tanzania . None ofA major strength of this study was its large sample size , which enabled the detection of an effect of SNPs on antibody levels even at a significance level adjusted for multiple testing. The sample size also allowed adjustment for a multitude of potential confounding factors, and having participants from ten sites in seven different countries sampled using standardized methodologies allowed an analysis for consistency of SNP effect across sites. This study finds an association of HbS (rs334) carriage with lowered antibody levels to merozoite antigens AMA1, MSP1 and MSP2, that is highly significant and consistent across study sites. This study demonstrates the feasibility of combining data from heterogeneous sources and the findings support the notion that genetic factors can determine an individual’s immune response to malaria.