Malaria epidemiology in Mauritania has been characterized on the basis of epidemiological strata, defined by climatic and geographic features, which divide the country into three zones: Sahelian zone, Sahelo-Saharan transition zone, and Saharan zone. The association between geographic stratification and malaria transmission was assessed through a series of parasitological and entomological surveys.Surveys were conducted during the ‘cool’ dry season in 2011, ‘hot’ dry season in 2012, and rainy season in 2013 in a total of 12 sentinel sites. Finger-prick capillary blood samples were collected from children aged 2–9 years old in randomly selected households for microscopic examination and rapid diagnostic test for malaria. Adult mosquitoes were sampled by pyrethrum spray catch and CDC light traps and identified using morphological keys and molecular tools.Plasmodium falciparum , Plasmodium vivax , P. falciparum-P. vivax , Plasmodium ovale , and Plasmodium malariae . A large majority of P. falciparum infections were observed in the Sahelo-Saharan zone. Malaria prevalence (P < 0.01) and parasite density (P < 0.001) were higher during the rainy season (2013), compared to cool dry season (2011). Plasmodium vivax was mainly observed in the Saharan region [43 of 59 (73 %) P. vivax infections], mostly in Nouakchott districts, with no significant seasonal variation. Of 3577 mosquitoes captured, 1014 (28.3 %) belonged to Anopheles spp. Anopheles gambiae was the predominant species in all three epidemiological strata during the ‘cool’ dry season in 2011 but was absent in all study sites, except for Teyarett district in Nouakchott, during the ‘hot’ dry season in 2012. During the rainy season in 2013, An. gambiae, Anopheles arabiensis, Anopheles pharoensis, and Anopheles rufipes were abundant in different zones.Of 3445 children included, 143 (4.15 %) were infected with malaria parasites including The results of the present study support the stratification of malaria in Mauritania. However, the Sahelian zone had the lowest malaria prevalence, while the Sahelo-Saharan zone had the highest malaria burden. Local changes due to anthropogenic factors  should be considered in order to optimize the control strategy. Plasmodium falciparum is endemic  and the Saharan zones . The results obtained during the ‘hot’ dry season in 2012 showed either a very low malaria infection rate  in the Saharan zone or a total absence of detectable blood-stage malaria parasites in the Sahelian and Sahelo-Saharan zones, suggesting an apparent interruption of malaria transmission in these latter two zones during the ‘hot’ dry season.In the Sahelian zone, malaria-positive smears were found in 6 of 350 (1.71 %) children during the ‘cool’ dry season in 2011 and in 8 of 337 (2.37 %) children during the rainy season in 2013  and Saharan zones  were much higher (P < 0.05). In the Sahelo-Saharan zone, the majority of children were infected with P. falciparum , followed by P. vivax , and P. ovale . Mixed P. falciparum-P. vivax infections  were also observed. Within the Sahelo-Saharan zone, most P. falciparum infections were observed near the border with Mali (P = 0.04) where war refugees have been resettled temporarily and where transmission was apparently interrupted during the hot season in 2012.The analysis of data by epidemiological strata showed that the Sahelian zone along the Senegal River was characterized by the lowest number of malaria-positive slides  during the study period , and only one child  was infected with P. falciparum. In the Sahelo-Saharan and Saharan zones, P. malariae was not found in the present study.By contrast, in the Saharan zone, the majority of children were infected with P. falciparum infections in Sahelo-Saharan zone, the median parasite density in 2013  was significantly higher (P = 0.018) than that observed in 2011   and 2013  (P = 0.143).The overall geometric mean parasite density  and median of malaria-infected children n = 143) were 804 asexual parasites/µl  and 820 asexual parasites/µl, respectively. Parasite densities found in three epidemiological strata are summarized in Table 3 were 80Anopheles spp. 1631 (45.6 %) Culex spp. and 932 (26.1 %) Aedes spp. Anopheles spp. (40 %) predominated over Culex (32 %) and Aedes (28 %) in the Sahelian zone during the ‘cool’ dry season in 2011. The proportions of Anopheles and Culex were similar (41.6 vs 43.9 %) during the 2013 rainy season in the Sahelo-Saharan transition zone. During the ‘hot’ dry season in 2012, the lowest number of mosquitoes was captured, and Anopheles spp. were absent in all study sites, except for Teyarett district in Nouakchott where Anopheles gambiae s.l. (n = 8) and Anopheles pharoensis (n = 1) were observed. In the two localities situated in northern Saharan zone, Akjoujt and Aoujeft, Anopheles mosquitoes were rarely captured during the entire study period.A total of 3577 mosquitoes were captured in randomly selected households in the 12 surveillance sites in 2011–2013. The numbers of mosquitoes found in each study site during different seasons are summarized in Table Anopheles specimens collected during the study period consisted of at least six species , Anopheles rufipes , An. pharoensis , Anopheles domicola , Anopheles ziemanni , and Anopheles rhodesiensis . Boghé and Gouraye in the Sahelian zone and Kankossa and Kobeni in the Sahelo-Saharan zone recorded the highest number of Anopheles mosquitoes caught during the ‘cool’ dry season in 2011 and during the rainy season in 2013, respectively. Overall, mosquitoes from the An. gambiae s.l. complex were the most abundant and widely distributed. During the ‘cool’ dry season in 2011, An. gambiae s.l. mosquitoes were predominant in all three epidemiological strata. During the ‘hot’ dry season in 2012, An. gambiae s.l. mosquitoes were captured only in Teyarett district, Nouakchott. Molecular identification of the specimens collected during the 2013 rainy season revealed that An. gambiae sensu stricto and An. arabiensis occur in sympatry in most sites in the sahelo-Saharan and Saharan zones. In the Sahelian zone, An. rufipes appeared as the predominant species in indoor collections during the rainy season, followed by mosquitoes from the An. gambiae complex. An. pharoensis was also widespread in all three zones during the rainy season.The es Table : An. gamThe present study is one of the most extensive epidemiological studies so far conducted in Mauritania. The survey covered 12 localities in nine administrative regions situated in three different epidemiological strata and included both parasitological and entomological data collection in 2011–2013. Despite the fact that the present survey was limited to children under nine years of age, previous studies on febrile patients spontaneously consulting at health centres have suggested that children aged between five and nine years represent the age group that is most affected by uncomplicated malaria and are the priority target population for malaria interventions , 17. ConP. falciparum monoinfections. Data reported in the present study from the Sahelian zone are in agreement with the results of a previous study in which health centres in Keurmassen and Boghé reported the lowest proportions of positive blood smears among febrile adult and paediatric patients during the rainy season (July–October 2010) and ‘cool’ dry season (December 2010–March 2011), as compared to febrile patients consulting health centres in Sahelo-Saharan  and Saharan  zones [P. falciparum , while seven of 39 patients had P. ovale or P. malariae. None of the febrile patients had P. vivax.In the Sahelian zone along the Senegal River, malaria burden in children two to nine years of age was relatively low, compared to other epidemiological strata, and was mostly due to t) zones . PositivP. falciparum, and 9 of 1040 (0.9 %) asymptomatic schoolchildren were malaria parasite carriers. The situation was similar on the other side of the border formed by the Senegal River, where malaria incidence has been low since the 1990s [Malaria prevalence was also shown to be low in Rosso , a city situated along the Senegal River, and its surrounding villages, in 2004–2006 . In thathe 1990s .P. falciparum malaria. The low frequency of malaria-positive smears observed in the Sahelian zone may be at least partly attributed to an effective implementation of a set of intensive priority interventions in this zone over the past years with the support of Global Fund, including the distribution of long-lasting insecticide-impregnated nets (LLIN), intermittent preventive treatment during pregnancy, and prompt treatment with artemisinin-based combination therapy (ACT) [In the Sahelian zone along the Senegal River, transmission was seasonal, with an apparent absence of patent parasitaemia during the ‘hot’ dry season. These findings were somewhat unexpected because of long-standing a priori assumptions that the Mauritanian side of the Sahelian zone bordering Senegal was thought to be highly endemic for py (ACT) . During P. falciparum monoinfection was predominant in the Sahelo-Saharan zone, but P. vivax monoinfection and P. ovale were responsible for close to one-fourth of malaria infections. Moreover, P. falciparum-P. vivax mixed infections  were observed only in the transition zone. Similar proportions of different Plasmodium species  were reported among 222 malaria-infected febrile patients consulting health centres in Kobeni, Kankossa, and Tamchekett in 2010–2011 [Malaria occurred more frequently in children in the Sahelo-Saharan transition zone, as compared to the Sahelian zone, during both the ‘cool’ dry season just after the rainy season in November–December 2011 as well as during the rainy season in October 2013. As in the Sahelian zone, there was no malaria-infected child in June 2012, suggesting an interruption of malaria transmission during the ‘hot’ dry season. In an earlier study in the Sahelo-Saharan zone, smear-positive febrile adult and paediatric patients  were mostly observed during the rainy season (July–October 2010), but smear-positive patients were also identified during the ‘cool’ dry season  and hot dry season  . The app010–2011 .Nbeika and Tamcheket are situated near the same latitude as Nouakchott, and both have permanent or semi-permanent artificial lakes. Compared to Kobeni and Kankossa, two other study sites in the Sahelo-Saharan zone, the levels of malaria transmission were much lower in these two sites with a relatively small population of less than 2000 residents. The highest malaria transmission levels observed in this zone were during the rainy season in 2013 in Kobeni and Kankossa, both of which are situated near the border with Mali. These border areas, in particular Kobeni, are associated with considerable human migration  and mass refugee flows. The mobility of parasite populations is facilitated in such circumstances, favouring the maintenance of malaria transmission or its re-emergence . Human pP. falciparum-infected symptomatic patients of all ages. An adequate number of patients fulfilling the inclusion criteria  was recruited in less than three weeks, confirming the relatively high level of malaria transmission in these areas during the rainy season.In another recent study conducted in Kobeni and Timbedra  during the rainy season in September–October 2013 [These findings in the Sahelo-Saharan zone are related with inadequate human resources, poor health infrastructure, and inadequate delivery of interventions, including insufficient supplies of RDT, ACT, sulfadoxine-pyrimethamine, and LLIN. Data on malaria interventions and quantities of essential drugs and preventive measures delivered to the zone are incomplete. However, available data from 2011–2013 strongly suggest that public health sector in the Sahelo-Saharan transition zone received much less quantities of intervention measures, as compared to the Sahelian zone. Preventive measures, such as LLIN and sulfadoxine-pyrimethamine for pregnant women, were not provided regularly, or even not provided at all to some study sites in the zone during the study period. In 2011 and 2012, RDT was either unavailable or its quantity was insufficient, but in 2013 the available quantity of RDT was sufficient to cover the needs in the Sahelo-Saharan zone. The quantity of ACT did not cover the needs of the population in the zone during the entire study period.P. vivax. Even during the dry season, there were children infected with P. vivax in Teyarett district in Nouakchott. These findings are in agreement with several studies conducted among febrile, symptomatic paediatric and adult patients presenting spontaneously at the Teyarett health centre, which showed an increasing incidence of P. vivax malaria and a decrease in P. falciparum-to-P. vivax proportions during recent years [P. vivax malaria occurs throughout the year among febrile residents consulting the Teyarett health centre and that the peak transmission period extends from October to December [P. vivax malaria also occurs in febrile patients residing elsewhere in the capital city of Nouakchott, as in Darnaim district and several other districts [P. vivax-susceptible human population, the presence of Anopheles mosquitoes, and man-made environmental modifications that favour the creation of larval habitats in Nouakchott [In the Saharan zone, malaria burden among children aged two to nine years old was lower than that of the Sahelo-Saharan transition zone but higher than that of the Sahelian zone. In sharp contrast to the Sahelian and Sahelo-Saharan zones, malaria transmission in the Saharan zone was characterized by a large predominance of nt years , 17, 18.istricts . These fuakchott , 9, 25. P. vivax was confirmed in Nouakchott and Aoujeft in the present study. Malaria was not found in Akjoujt in the present study. The apparent absence of malaria transmission in Akjoujt is in agreement with the absence of Anopheles mosquitoes. However, in a year-long monitoring in Akjoujt health centre, 22 malaria-infected patients were found among 467 symptomatic patients spontaneously consulting the health centre [P. vivax during the rainy season and ‘cool’ dry season, respectively. In Atar , the regional capital of Adrar region that is located near but further north than Akjoujt and Aoujeft in the Saharan desert, P. vivax-infected symptomatic patients have been diagnosed using microscopy and rapid diagnostic test, confirmed using PCR, and successfully treated with chloroquine in 2013 [P. vivax in Atar is at least partly related to frequent visits of some Nouakchott residents to Atar , especially during the rainy season when some districts of the capital city are often flooded due to lack of water evacuation system. At present, available data suggest that P. vivax transmission in Aoujeft and Akjoujt appears to occur at a low level, possibly due to low human and Anopheles mosquito population densities. These data suggest that P. vivax could be more widely spread in Mauritania than currently known and possibly extend from the Sahelo-Saharan transition zone along the southern border with Mali  to as far north in the Saharan desert as Aoujeft, Akjoujt, and Atar, including the capital city, Nouakchott [The presence of h centre . In that in 2013 . The preuakchott , 18, 26.P. vivax was not found in the Sahelian zone along the Senegal River. In an earlier study performed in Rosso [P. vivax was diagnosed in seven symptomatic patients during the dry season in February 2004. However, the authors have suggested that it may be doubted whether local P. vivax transmission actually occurs in Rosso since the majority (5 of 7 patients) had a recent travel history to Nouakchott. Further surveys are warranted, particularly along the Senegal River, in the eastern regions, and oases in the northern regions to establish the current geographic limits of P. vivax transmission in Mauritania. The consequences of regional and seasonal human migratory movements, including nomads, migrants, and refugees fleeing armed conflicts in neighbouring countries, have not been evaluated in terms of health risks and may need to be taken into consideration to understand P. vivax distribution in the country.In the present study, in Rosso , P. vivaAnopheles mosquitoes in these zones during the ‘hot’ dry season in 2012. Since the present survey was not conducted continuously in time, further surveillance is needed to determine monthly malaria incidence in these strata. In the Saharan zone, the low frequency of P. vivax malaria (3 of 348 children) observed in Teyarett, Nouakchott, but not in any other sentinel sites, during the dry season is compatible with a permanent presence of Anopheles mosquitoes in the capital city where numerous artificial larval habitats, including potable water leaks and standpipes, exist all year round [The interruption of malaria transmission, as suggested by the complete absence of detectable blood-stage malaria parasites in the Sahelian and Sahelo-Saharan zones, is in agreement with the absence of ar round , 8. Nonear round .An. gambiae s.l., which was the most abundant species found in almost all localities, An. pharoensis, and An. funestus. Molecular analysis of the An. gambiae complex showed the predominance of An. arabiensis. Although mosquitoes were collected from different surveillance sites in the Sahelian and Sahelo-Saharan zones, the results of the previous study are in general agreement with those of the present study. However, more longitudinal studies are required to identify and characterize malaria vectors involved in P. falciparum and P. vivax transmission in different epidemiological strata in Mauritania.There is only one recent published entomological survey on the distribution of malaria vectors in Mauritania . In thatPlasmodium spp. and Anopheles spp. in different parts of the country and delimit malaria transmission zones with the aim to mobilize available resources and commit funds to appropriate areas for an effective malaria control programme.The results presented in this study do not reflect the prevalence of malaria in the general population in Mauritania, nor do they intend to provide a detailed distribution map of its vector mosquitoes throughout the country. The target population consisting of children between two and nine years of age and discontinuous surveys during the three-year period are major limitations of the present study. However, the present study is one of the first steps towards a more comprehensive survey to map the current distribution of Although the malaria prevalence rate cannot be calculated from the data reported here, the present study provides some indications for further epidemiological work in the country. Moreover, the artificial division of the country into three epidemiological strata based on geographic and climatic features to analyse the current malaria situation provides a useful starting framework but should be complemented by taking into account other parameters related to human activities and cross-border as well as internal population movement.P. falciparum-associated morbidity and mortality more effectively, more resources should be made available in the Sahelo-Saharan transition zone, where the problem of malaria is compounded by population movement  and the presence of war refugees. P. vivax malaria is a growing public health problem which is spreading in previously malaria-free zone in the Saharan desert, including the capital city where close to one-third of the country population is concentrated. Further studies are needed to define the geographic limits of P. vivax, especially in the northern and eastern parts of the country where surveys have rarely been conducted.The Sahelian zone along the Senegal River is characterized by relatively low malaria burden, compared to the other two epidemiological strata, due to malaria control interventions on both sides of the border with Senegal. To reduce


Plasmodium vivax was long considered to have a low mortality, but recent reports from some geographical areas suggest that severe and complicated vivax malaria may be more common than previously thought.Malaria caused by The primary objective of this systematic review and meta-analysis was to describe the reported clinical characteristics and the geographical variation in prevalence of reported severe vivax malaria and its change over time derived from English-language articles published since 1900. Medline and Scopus databases were searched for original papers on severe vivax malaria, using as inclusion criteria modified 2010 WHO criteria for the diagnosis of severe falciparum malaria. Articles before 1949 were identified through reference lists in journals, textbooks, and personal collections of colleagues.3) was the most common “severe” manifestation . The case fatality was 0.3% . Severity syndromes varied widely between different geographical areas, with severe anaemia being most prominent in areas of high transmission and chloroquine resistance.A total of 77 studies with reported severe vivax malaria and 63 studies with no reported severe vivax malaria  were included. The 77 studies with reported severe vivax malaria were mainly from India (n = 33), USA (n = 8), Indonesia (n = 6), and Pakistan (n = 6). Vivax endemic countries not reporting severe vivax malaria beyond individual case reports included: the Greater Mekong Sub-region, China, North Korea, Bangladesh, Afghanistan, Middle East (except Qatar), the horn of Africa, and Madagascar. Only 17/77 reports were from before 2000. Vivax mono-infection was confirmed by PCR in 14 studies and co-morbidities were ruled out in 23 studies. Among the 77 studies reporting severe vivax malaria, severe thrombocytopenia  contains supplementary material, which is available to authorized users. Plasmodium species causing disease in humans, Plasmodium falciparum is the main cause of severe and fatal disease. Traditionally Plasmodium vivax has been considered relatively benign, except for occasional severe manifestations notably severe anaemia and the acute respiratory distress syndrome [P. vivax infections has been recognized for over a century, the last decade has seen a remarkable increase in case reports, series and studies describing severe and fatal vivax malaria. An estimated 2.8 billion people globally live in one of the 95 countries endemic for P. vivax[P. vivax endemic areas transmission is low and seasonal , the Americas, and the horn of Africa (and Madagascar); whereas the rest of Africa is relatively spared because of the absence of the Duffy blood group which mediates parasite invasion [Of the five syndrome . AlthougPlasmodium vivax was associated with a 7.7% mortality in the malariatherapy of neurosyphilis, but this was attributed in part to the debilitated condition of the patients and mortality in Plasmodium malariae infections was even higher [P. vivax infection, severe disease and death than recognized previously [P. vivax infection. The primary objective was to describe the geographical variation in prevalence of severe vivax malaria and the different presenting syndromes and characteristics, as well as to analyse the changes in reporting over time.Occasional fatalities in vivax malaria have been reported since the species was first recognized, particularly in already debilitated patients, and significant mortality has been attributed to vivax malaria in the first and second world wars although details of speciation are often scanty. n higher . Early pn higher  and smaln higher , 11, buteviously –16. A syEligible studies on severe vivax malaria since 1949 were identified in Medline (via PubMed) and Scopus (via Scopus). The final search and updates in both databases were carried out on January 28, 2014. Articles before 1949 (Pre-PubMed era) were mainly searched through other sources including reference lists of published journal articles and (old) textbooks and through gifts of personal collections of colleagues. Details on variables, search terms, and search strategies are explained in Additional file 3) [P. vivax mono-infection were selected. Studies included those where the denominators were both inpatients and outpatients with vivax malaria and studies reporting only inpatients with vivax malaria patients. Exclusion criteria included duplicate reporting, mixed infections (P. vivax with any other Plasmodium species), and insufficient data for extraction. All types of study designs with primary data (having patients of either gender or any age) were eligible, but case reports and case series were not included in pooled calculations of prevalence because the denominator was uncertain. The complete selected articles were analysed and data were coded into a data extraction form (DEF).All literature in English describing clinical studies in human vivax malaria was considered, recognizing that there is also a substantial literature in other languages. Malaria was diagnosed by peripheral blood smear (PBS) and in recent years by rapid diagnostic test (RDT) or polymerase chain reaction (PCR). Since severity criteria for severe vivax malaria have not been specified yet, modified criteria defined in the 2010 WHO supplement on severe falciparum malaria were used as the outcome of interest used for inclusion, with the addition of thrombocytopenia as this has been used as a severity criterion in several publications   [see AddEligibility for inclusion of every article was reviewed by independent reviewers . Disagreements between reviewers were resolved by discussion, and extracted data were validated by an independent person (Watcharee Chokejindachai).For the meta-analysis, pooled prevalence was calculated aswhere i = Prevalence of the severity sign in each study;pi =1/var (pi), which was the weight of each study.wThe pooled prevalence and 95% CI of each severity sign was calculated only if it was reported in at least three reports using the following command in STATAwhere n = Total number of patients with a specific severity sign due to vivax malaria; freq = Total number of patients with vivax malaria in the study.2 test. Software STATA version 12  was used for statistical analysis.A subgroup analysis was performed in order to compare prevalence of severity signs before and after 2000 and between different WHO regions  and countries, using ChiInitially 274 and 412 studies were identified in the Medline and Scopus databases, respectively. Of these, 289 were duplicates and 278 were ineligible for other reasons, including: only case reports and case series (n = 71), non-English language (n = 41), and disease caused by obvious co-morbidities (n = 8)  occurred 23 times less frequently than that seen with falciparum malaria and was estimated as occurring in one in 29,500 infections [P. vivax (not PCR-confirmed) was one in 858 infections, with the risk being 15.2 fold less than that with P. falciparum[In Indonesian Papua, coma associated with PCR-confirmed fections . In Thaialciparum. Mortalialciparum.Among 77 studies with severe vivax malaria patients, 62 studies reported severity signs in both inpatients and outpatients of vivax malaria. In these studies, 31 reported cerebral malaria, six generalized seizures, 14 renal dysfunction, 19 respiratory dysfunction, 31 hepatic dysfunction, 17 abnormal bleeding/DIC, seven haemoglobinuria, eight hypoglycaemia, four metabolic acidosis, six circulatory collapse/shock, 23 severe anaemia, 21 severe thrombocytopenia, and 28 studies reported fatal cases [see Additional files The pooled prevalence of the various severity signs in studies that reported both inpatients and outpatients of vivax malaria are shown in Additional file Among 77 studies with severe vivax malaria patients, 15 studies described severity signs only in inpatients vivax malaria. In these studies, 11 reported cerebral malaria, 5 repeated generalized seizures, 6 renal dysfunction, 8 respiratory dysfunction, 12 hepatic dysfunction, 7 abnormal bleeding/DIC, 3 haemoglobinuria, 6 hypoglycaemia, 4 metabolic acidosis, 5 circulatory collapse/shock, 10 severe anaemia, 6 severe thrombocytopenia, and 9 studies reported deaths [see Additional files The pooled prevalence of severity signs among reported inpatients with vivax malaria are shown in Additional file Pf:Pv ratio for all signs of malaria severity. Highest mean Pf:Pv ratio was present for fatalities (4.0), followed by repeated generalized seizures (3.64), renal dysfunction (2.85), hypoglycaemia (2.75), and cerebral malaria (2.73). Lowest mean Pf:Pv ratio was for severe thrombocytopenia (1.19) and respiratory dysfunction (1.37).Figure P. vivax infections, (which in most studies was confirmed as mono-infection by PCR). Of these 33 (42.9%) studies came from India. In contrast, a large number of endemic countries do not report severe vivax malaria, including the Greater Mekong Sub-region, North-east China, North-Korea, Bangladesh, Afghanistan, Middle East (except Qatar), Somalia, and Madagascar. There are two general presentations reported; severe anaemia in young children from the island of New Guinea where transmission is high, and vital organ dysfunction usually in adults from low transmission settings. A large variation in organ involvement was observed in the latter group. Vivax malaria was reported as an important cause of hospital admission in endemic areas, but a major problem in determining the true incidence and prevalence of severe vivax malaria is that severe disease has been considered relatively unusual in vivax malaria, and so its absence from a clinical series has not been considered noteworthy. For example in over 1,000 patients with severe malaria admitted to a centre in Ho Chi Minh city, Vietnam, specializing in its management there was one case of severe vivax  malaria and no cases of vivax malaria associated acute renal failure , but this paucity of severe disease was not considered noteworthy and so not reported. This contrasts with a prevalence of severe disease amongst admitted cases of over 30% in some series. Thus estimating incidence and prevalence from series which do report severe vivax malaria provides overestimates. Furthermore the most prevalent severity sign reported was thrombocytopenia <50,000/μL (26.1%), which is not regarded as a severity sign in falciparum malaria, and has not been validated as an independent severity measure in vivax malaria. The least prevalent manifestation was haemoglobinuria (0.1%). In studies with only inpatients of vivax malaria, thrombocytopenia (35.9%) was the most commonly reported severity sign, while least prevalent severity sign was respiratory dysfunction (1%). Mortality in studies with only inpatients of vivax malaria was 28.2%, compared to 0.2% in groups with both inpatients and outpatients of vivax malaria.There is substantial variation in the reported geographic distribution and the incidence of severe manifestations of vivax malaria. This analysis also shows a large increase since the year 2000 in the number of studies and numbers of patients reported with severe P. vivax, compared to 93/2664 (3.5%) with P. falciparum or mixed infection [P. vivax compared to 64/382 (17%) with P. falciparum or mixed infection [It seems unlikely that underreporting can explain the scarcity of cases in the last century, although there are only few data to support this. A study in American and Allied soldiers in India reported in 1944, described cerebral malaria in 5/1,375 cases (0.4%) with nfection . An Indinfection . A more nfection . One conP. vivax malaria (3 out of 2573 patients) [P. vivax-related pulmonary oedema has been reported from this area [The geographical heterogeneity is more substantiated by data; a study from the Thai-Myanmar border from 1997 describes a very low incidence of patients with severe his area .P. vivax and P. falciparum, respectively. In the same population patients with both falciparum and vivax malaria and thrombocytopenia <50,000/mm3 had an increased mortality, although as not all the other clinical and laboratory measures used to assess disease severity were recorded it is unclear whether thrombocytopenia was an independent risk factor or not [Chloroquine resistance has been well documented on the Island of New Guinea  where many of the Indian reports on severe malaria originate, the population prevalence of vivax malaria is only around 1% . In contrast, the population prevalence is higher in endemic regions Myanmar , 38, whinfection –41. Of tnozoites . In manynozoites , 43, 44.nozoites .P. vivax parasitaemia prevalence is documented and there is proper evaluation of other concomitant illnesses. One such study from Papua, Indonesia, comparing population prevalence with coma in hospitalized patients estimated the incidence of cerebral malaria as 1:29,486 in P. vivax compared to 1:1,276 in P. falciparum infections. This suggested that P. vivax-associated coma is 23 times less common than P. falciparum-associated coma [Further detailed study of the reported severe manifestations might shed some light on pathogenesis. In Bikaner, where severe vivax malaria is commonly reported, high rates of acute kidney injury are reported in severe vivax malaria in children aged 0–5 years (8/41: 19.5%), an age group in whom acute tubular necrosis  is rare. Acidosis was reported in 7/65 children with vivax malaria  but its aetiology was unclear. In falciparum malaria acidosis is associated with a high lactate-pyruvate ratio indicating an anoxic pathogenesis, whereas in sepsis lactic pyruvate rations are not elevated. There is clearly a need for more in-depth prospective epidemiological studies, where background age-stratified population ted coma .Epidemics of severe vivax malaria were reported from the USSR over fifty years ago. Changes in strain virulence could explain the recent and geographical restricted increase in reported cases, although there are no hard data available to support this hypothesis. Vivax strains clearly do differ between regions, which is most evident in the observed relapse patterns. Pathophysiological studies exploring difference in parasite virulence could reveal whether more virulent strains of this parasite with a once benign reputation have appeared.An earlier systematic review on complicated vivax malaria in South America was reported by Lacerda and colleagues in 2012 . In thisPlasmodium infection. Another shortcoming is that we only selected English language articles, whereas there is a significant literature in other languages, e.g. reports from Amazonia are often in Portuguese.This analysis had several limitations. The pooled prevalence estimates of severe manifestations are based only on reported studies which include cases of severe vivax malaria and this provides a selection bias. Absence of severe disease amongst patients with vivax malaria appears much less likely to be reported. Assessing the pooled prevalence of severe vivax malaria against the general population or all vivax malaria cases as denominator is not feasible with the current data. Nearly all the estimated worldwide 390 million clinical cases of vivax malaria/year  are not P. vivax endemic world, which cannot be explained with the current understanding of the disease and warrants further study into its aetiology. The use of thrombocytopenia as an independent severity criterion requires justification. In addition, more detailed epidemiological studies are needed, which adequately exclude co-morbidities, and take into account transmission intensity and anti-malarial drug susceptibility. As for severe P. falciparum malaria, P. vivax-specific criteria for defining severe disease are necessary to facilitate comparison between studies and establish its true prevalence.In summary, there has been a marked increase in reported cases of severe vivax in certain geographical regions of the Variables, search terms, and search strategy used in this study.(DOCX 24 KB)Additional file 1:Definitions for the diagnosis of severe vivax malaria in this study.(DOCX 24 KB)Additional file 2:Characteristics of the 77 studies with reported severe vivax malaria patients.(DOCX 97 KB)Additional file 3:Detailed analysis of pooled prevalence of severity signs among reported severe vivax malaria patients (77 studies).(DOCX 38 KB)Additional file 4:Prevalence of cerebral malaria among both outpatients and inpatients of vivax malaria.(DOCX 51 KB)Additional file 5:Prevalence of repeated generalized seizures among both outpatients and inpatients of vivax malaria.(DOCX 27 KB)Additional file 6:Prevalence of renal dysfunction among both outpatients and inpatients of vivax malaria.(DOCX 39 KB)Additional file 7:Prevalence of respiratory dysfunction among both outpatients and inpatients of vivax malaria.(DOCX 40 KB)Additional file 8:Prevalence of hepatic dysfunction among both outpatients and inpatients of vivax malaria.(DOCX 51 KB)Additional file 9:Prevalence of abnormal bleeding/DIC among both outpatients and inpatients of vivax malaria.(DOCX 39 KB)Additional file 10:Prevalence of haemoglobinuria among both outpatients and inpatients of vivax malaria.(DOCX 29 KB)Additional file 11:Prevalence of hypoglycaemia among both outpatients and inpatients of vivax malaria.(DOCX 30 KB)Additional file 12:Prevalence of metabolic acidosis among both outpatients and inpatients of vivax malaria.(DOCX 28 KB)Additional file 13:Prevalence of shock among both outpatients and inpatients of vivax malaria.(DOCX 29 KB)Additional file 14:Prevalence of severe anaemia among both outpatients and inpatients of vivax malaria.(DOCX 219 KB)Additional file 15:Prevalence of severe thrombocytopenia among both outpatients and inpatients of vivax malaria.(DOCX 40 KB)Additional file 16:Prevalence of death among both outpatients and inpatients of vivax malaria.(DOCX 52 KB)Additional file 17:Pooled prevalence of severity signs among both inpatients and outpatients of vivax malaria (62 studies).(DOCX 24 KB)Additional file 18:Prevalence of cerebral malaria among only inpatients of vivax malaria.(DOCX 34 KB)Additional file 19:Prevalence of repeated generalized seizures among only inpatients of vivax malaria.(DOCX 29 KB)Additional file 20:Prevalence of renal dysfunction among only inpatients of vivax malaria.(DOCX 29 KB)Additional file 21:Prevalence of respiratory dysfunction among only inpatients of vivax malaria.(DOCX 32 KB)Additional file 22:Prevalence of hepatic dysfunction among only inpatients of vivax malaria.(DOCX 36 KB)Additional file 23:Prevalence of abnormal bleeding/DIC among only inpatients of vivax malaria.(DOCX 31 KB)Additional file 24:Prevalence of haemoglobinuria among only inpatients of vivax malaria.(DOCX 26 KB)Additional file 25:Prevalence of hypoglycaemia among only inpatients of vivax malaria.(DOCX 29 KB)Additional file 26:Prevalence of metabolic acidosis among only inpatients of vivax malaria.(DOCX 28 KB)Additional file 27:Prevalence of shock among only inpatients of vivax malaria.(DOCX 29 KB)Additional file 28:Prevalence of severe anaemia among only inpatients of vivax malaria.(DOCX 34 KB)Additional file 29:Prevalence of severe thrombocytopenia among only inpatients of vivax malaria.(DOCX 29 KB)Additional file 30:Prevalence of death among only inpatients of vivax malaria.(DOCX 34 KB)Additional file 31:Pooled prevalence of severity signs among only inpatients of vivax malaria (15 studies).(DOCX 24 KB)Additional file 32:


In 2004, Ghana implemented the artemisinin-based combination therapy (ACT) policy. Health worker (HW) adherence to the national malaria guidelines on case-management with ACT for children below 5 years of age and older patients presenting at health facilities (HF) for primary illness consultations was evaluated 5 years post-ACT policy change.Cross-sectional surveys were conducted from 2010 to 2011 at HFs that provide curative care as part of outpatient activities in two districts located in the middle belt of Ghana to coincide with the periods of low and high malaria transmission seasons. A review of patient medical records, HW interviews, HF inventories and finger-pricked blood obtained for independent malaria microscopy were used to assess HW practices on malaria case-management.Data from 130 HW interviews, 769 patient medical records at 20 HFs over 75 survey days were individually linked and evaluated. The majority of consultations were performed at health centres/clinics (68.3 %) by medical assistants (28.6 %) and nurse aids (23.5 %). About 68.4 % of HWs had received ACT-specific training and 51.9 %, supervisory visits in the preceding 6 months. Despite the availability of malaria diagnostic test at most HFs (94 %), only 39.8 % (241) out of 605 (78.7 %) patients who reported fever were investigated for malaria. Treatment with ACT in line with the guidelines was 66.7 %; higher in <5 children compared to patients ≥5 years old. Judged against reference microscopy, only 44.8 % (107/239) of ACT prescriptions that conformed to the guidelines were “truly malaria”. Multivariate logistic regression analysis showed that HW were significantly more likely to comply with the guidelines if treatment were by low cadre of health staff, were for children below 5 years of age, and malaria test was performed.Although the majority of patients presenting with malaria received treatment according to the national malaria guidelines, there were widespread inappropriate treatment with ACT. Compliance with the guidelines on ACT use was low, 5 years post-ACT policy change. The Ghana NMCP needs to strengthen HW capacity on malaria case-management through regular training supported by effective laboratory quality control measures. Significant progress has been made in the last decade towards achieving global malaria targets although the disease burden is still relatively high in sub-Saharan African countries, especially among children <5 years of age , 2. AccoPlasmodium resistance across Africa [Prompt and effective malaria case management has been the main control strategy , 4. Ghans Africa . The ants Africa . The sucs Africa , 8. The s Africa , 10.Ghana revised her national guidelines for malaria case management to address changes in the anti-malarial drug policy . The polRestricting treatment to parasitological diagnosis of malaria among older children and adults is expected to reduce over-diagnosis of malaria and inappropriate use of relatively expensive ACT in the treatment of fevers . Proper -ACT policy implementation by evaluating health worker (HW) compliance to the national malaria guidelines on ACT prescriptions.In this study, the quality of uncomplicated malaria case management for children <5 years and older patients presenting for primary illness consultation was assessed at health facilities (HFs) in two districts of the middle belt of Ghana 5 years post2 and has a resident population of 134,970 as at 2009 [The study was conducted in Kintampo North and South Districts, which lie within the forest savannah transitional ecological zone in the middle belt of Ghana. The Kintampo districts cover an area of 7162 km at 2009 . Subsist at 2009 . Like in at 2009 . License at 2009 . There aThe design was a cross-sectional HF survey involving review of patient medical records, HW interviews, HF inventory and independent blood slide reading by a reference laboratory to assess provider compliance to malaria case-management based on the Ghana national malaria treatment guidelines.-class correlation 0.2 to be able to detect an assumed lesser compliance of ≤53 % in this non-controlled study with 95 % confidence and with 90 % statistical power. A cluster was defined as all patient consultations performed at a HF on 1 day during regular working hours from 8 am to 5 pm. A primary illness consultation was defined as the first time a patient visited the HF for the treatment of the presenting illness episode.In a controlled trial carried out in southern Ghana compliance to malaria management with ACT based on malaria RDT and blood slide was 60.1 and 57.1 %, respectively . Seven hThree cross-sectional cluster surveys each lasting 5–8 weeks were conducted in January 2010, June 2010 and in January 2011 at HFs that provide curative care as part of outpatient activities. These surveys were planned to coincide with the periods of low (January 2010 and January 2011) and high malaria transmission seasons (June 2010). Data collection was undertaken by a team comprising of a clinician, a laboratory technician and field data collectors under the supervision of the study investigators.A list of 19 HFs in the study area that provided curative care as part of an out-patient setting during regular working hours from 8 am to 5 pm was used as the sampling frame. The HFs in the sampling frame increased to 20 with the establishment of an additional HF before the commencement of the second survey. Using a written programme in STATA, the HFs were randomly ordered and numbered consecutively. The HFs were assigned to pre-determined survey dates and visited in the same order and date as pre-determined. No other facility was visited on a survey day when the scheduled facility was found closed. HFs closed on the date of the survey were visited during a “catch-up period” on the same day of the week as originally determined. Dates of visits were not announced in advance to HF staff. On arrival at each HF, all HWs performing patient consultations were assigned identification numbers. Using a structured questionnaire, HWs were interviewed on their most recent ACT specific training, supervisory visits received and knowledge of the national malaria treatment guidelines. An inventory of diagnostic test kits, ACT and other medications available at the HF was made before patient consultations began.On a survey day, all patients at the HF who met the inclusion criteria of seeking primary consultation for their illness were asked to provide informed consent for participating in the study. After receiving treatment, patients who had given an informed written consent had their weight and axillary temperature re-assessed using standardized equipment by the survey team. Patient demographics, vital signs, medical records and insecticide treated net (ITN) use were recorded on a standardized questionnaire. About 0.5 µl of whole blood was collected into EDTA (ethylenediaminetetraacetic acid) tubes and stored in a cool box and transferred to the reference laboratory for malaria microscopy. A review of patient medical records was performed by the team leader to extract medical information of the care patient received on the survey day. Information extracted from the patient folders included HWs’ record of patient medical history, presenting symptoms, diagnosis and treatment.Thick and thin malaria films were prepared from blood collected and stained with Giemsa using the method recommended by WHO for blood smear preparation. The malaria blood smears were read independently by two experienced microscopists. In cases where the results were discrepant, a third microscopist read the slide for concordence with one of the two earlier readers. Assuming a count of 8000 leukocytes/dl of blood, parasite densities were calculated by counting the number of asexual parasites per 200 white blood cells [All data forms were checked manually for completeness and consistencies before data processing. Epi info version 7 software package was used for double entry, validation and verification of all data. Analysis was carried out using STATA, version 11 . The quality of outpatient malaria case management for each patient was evaluated by individually linking patient data  to the information of the HW and HF where patient received care on the survey day. The availability or absence of diagnostic test kits, medicines and ACT-specific training on the quality of care was further assessed.The primary outcome for assessment was provider compliance based on the case management guidelines of the Ghana NMCP otherwise referred to as appropriate ACT treatment . For allA comparative analysis of HW routine use and interpretation of malaria diagnostic test for <5 children and older patients with respect to a presentation of fever and ACT prescriptions was performed. The sensitivity, specificity as well as positive and negative predictive values (NPV) of routine diagnostic tests relative to the reference microscopy were determined.Univariate analysis was used to identify predictors of provider compliance to the national malaria treatment guidelines. Seasonality of malaria transmission and other explanatory variables such as age of patient, mode of diagnosis, type and level of HF, HW cadre and training on IMCI that showed a tendency of association with provider compliance were included in the multivariate logistic regression. Data was summarized as frequencies and proportions with corresponding 95 % confidence interval adjusted for clustering by HF.Ethical approval was provided by the Ghana Health Service Ethics Review Committee (GHS-ERC) and Kintampo Health Research Centre Institutional Ethics Committee (KHRC-IEC). Permission to conduct the HF surveys were obtained from the local health administrations. Informed written consent was obtained from patients and caretakers of children who were willing to participate in the study. Children aged 12–17 years of age were requested to provide their assent in addition to consent by parent or accompanying adult without which a patient was excluded from the study. Unaccompanied minors were excluded as per regulations of the KHRC-IEC. Informed written consent were also obtained from HWs who performed patient consultation on the day of survey. To maintain confidentiality, a unique study number/code was assigned to each participant as an identifier at enrolment for labelling all blood samples and study forms. All study data were stored securely with restricted access.Overall, 843 patient folder reviews and 132 HW interviews were performed at 20 HFs through 83 HF visits during three cross-sectional surveys  or had incomplete inventories (2) on the day of the visit. Seventy-four patient records were excluded from the analysis. Those excluded comprised of 18 patients who were on follow-up visits for previous illness and 36 pregnant women on the basis that the national guidelines does not recommend ACT treatment for pregnant women in the first trimester and the team did not plan for gestational assessment as part of this study. Others were 12 patients who did not consent to blood draw and 8 patients seen by two HWs whose interviews were incomplete. Analysis was, therefore, carried out on 769 patients’ data individually linked with each of the 130 HWs and 75 facility information of where patient received care on each survey day.The proportion of children <5 years of age presenting for a primary illness consultation was higher during the high malaria transmission season (HMTS) (33.8 %) than the low malaria transmission season (LMTS) 26.1 %)  were seen at the health centres/clinics  of patients who presented with fever had a malaria diagnostic test (RDT or microscopy) performed; 47.9 % in children <5 and 36.0 % in patients ≥5 years. Of patients with fever who had a malaria test performed, 70.2 % (167/238) had malaria positive results more commonly in <5 children (82.4 %) than older patients (62.6 %). Among the 164 patients who had no report of fever, 52 (31.7 %) had a malaria test performed of which 45.1 % (23/52) were positive; marginally higher in children <5 years (50 versus 44.2 %).The prescription of ACT for patients who had fever and malaria parasite was high , 93.3 % in children <5 years and 94.6 % in patients ≥5 years. Furthermore, presumptive treatment with ACT for febrile children <5 years was very high . Contrary to treatment recommendation, 159 (60.5 %) of patients ≥5 years who presented with fever were prescribed ACT without a malaria test performed.Among 164 patients presenting with no fever, 31.7 % (52/164) were tested. Of these, 45.1 % (23/52) were positive and 91.3 % (21/23) were treated. No treatment was given to those found negative.in <5 children, fever or a history of fever in the absence of other causes of fever should be considered malaria and treatment commenced immediately without waiting for laboratory results. Three febrile children whose malaria test were not documented were also excluded.In computing the primary outcome, 16 children <5 years who presented with fever but had a negative malaria test were excluded. This was done to prevent judging as incorrect HW practice of prescribing ACT for <5 children who had a negative malaria test as the national treatment guidelines states that Of the 750 patients presenting at HFs for primary illness consultation, 66.7 % were treated in accordance with the national malaria treatment guidelines; 239 received ACT whilst ACT were appropriately withheld in 261 patients. About 89.2 % (239/268) of <5 children presenting with at least fever and patients ≥5 years with uncomplicated malaria were correctly treated with ACT. ACT correctly withheld in both <5 children who had no fever and older patients who had no routine diagnosis of uncomplicated malaria was 54.2 % (261/482). Judged against results from the reference microscopy, however, only 44.8 % (107/239) of routinely prescribed ACT that conformed to the malaria guidelines were “truly malaria”. On the other hand, 8 % (21/261) of patients missed the opportunity to receive ACT during routine care due to either false negative results.The accuracy of the routine diagnostic tests namely malaria RDT and microscopy was determined with reference to expert microscopy Table . The senWhen univariate analysis was carried out, HW compliance in public HFs was found to be 66 % higher compared to private HFs. CHPS were 92 % more likely to conform to treatment guidelines compared to hospitals. Compared to medical doctors, less qualified HWs such as midwives, community health nurse (CHN) and nurse aids were 4.74 times ; 3.17 times  and 1.69 times  more likely to conform to treatment guidelines.Results of the multivariate logistic regression shows that mode of diagnosis for patients, age group and the cadre of HW were significantly associated with HW compliance to the national malaria treatment guidelines Table . A cliniIn this study, 66.7 % of patients presenting at HFs for primary illness consultation received treatment that was in line with the 2009 national malaria case-management guidelines 5 years after the deployment of the ACT policy in Ghana. Appropriate treatment with ACT was significantly higher in <5 children (85.9 %) than in older patients (59.7 %). The prescription of ACT based on a correct diagnosis of malaria as indicated by the national guidelines was high (89.2 %). However, 45 % of patients received ACT based on a wrong diagnosis of malaria. As a result of the low sensitivity of routine test, 8 % of patients who were confirmed by reference microscopy to have malaria missed the opportunity for any anti-malarial treatment.HWs non-compliance with the guidelines was a major cause of over-treatment with ACT. HW non-compliant behaviour observed in this study is similar to results from southern Ghana  and acroThe lower compliance with ACT treatment based on presumptive diagnosis with its attendant over-treatment justifies the transition to new guidelines by the WHO requiring parasitological diagnosis of malaria universally regardless of age and malaria transmission levels Table . UnfortuHW disregard to the existing guidelines raises questions about HW attitude and confidence in test results. For example, about 17 % of patients ≥5 years who presented with fever but had a negative test of malaria received ACT. Arguably, the unacceptably low sensitivity and NPV of routine diagnostics especially malaria microscopy compared to the reference microscopy could possibly explain the mistrust and low use of diagnostic tools. A study conducted in Tanzania corroborates these observations . The lonThe management of <5 children was more likely to conform to the guidelines compared to older patients Table . The higCHNs and midwives are low in the hierarchy of the HW professional ladder due to their limited scope of practice compared to medical officers. Their relatively huge presence seen in the management of most patients presenting for primary consultations notwithstanding their training on malaria case-management shows major task shifting, an indication of the shortage of medical doctors/medical assistants at the HFs in rural communities. The findings that lower cadre of health staff adhered much more closely to the guidelines compared to medical doctors as found in studies conducted in Kenya and rural Tanzania , 32 undeResults from this study also highlights the need for improved laboratory capacity at the primary care level for early and effective management of both malarial and non-malarial fevers. Under-diagnosis of malaria due to low sensitivity of routine diagnostics is a major concern with far-reaching clinical consequences. The “missed opportunity” for early treatment of malaria especially in children could result in severe malaria with high fatality outcome . LikewisThe interpretation of findings from this study should be interpreted with caution as Hawthorne effect could play an important role considering the context in which data was collected; issues related to observer effects, i.e. a positive change in the behavior of HW resulting in a better than usual performance when observed or monitored closely , 36. ThiAlthough a high proportion of <5 children presenting with fever and older patients with uncomplicated malaria received ACT in accordance with the national malaria guidelines, inappropriate treatment with ACT was still high especially among older patients five years after anti-malarial policy change. This was due to poor interpretation of the national malaria treatment guidelines particularly on malaria test result and low quality of routine malaria diagnostics.Concerted effort towards improving rational use of ACT by HWs is urgently needed if the anticipated benefits of the policy change are to be realized. The Ghana NMCP needs to strengthen the capacity of HWs through regular training  and supe


Plasmodium falciparum and occur in sub-Saharan Africa. One of the major complications asscociated with malaria is severe anaemia, caused by a cycle of haemoglobin digestion by the parasite. Anaemia due to falciparum malaria in children has multifactorial pathogenesis, which includes suppression of bone marrow activity. Recent studies have shown that haemozoin, which is a by-product of parasite haemoglobin digestion, may play an important role in suppression of haemoglobin production, leading to anaemia. In this study we correlated the levels of erythropoietin (EPO), as an indicator of stimulation of haemoglobin production, to the levels of monocyte acquired haemozoin in children with both severe and uncomplicated malaria. There was a significantly negative correlation between levels of haemozoin-containing monocytes and EPO, which may suggest that haemozoin suppresses erythropoiesis in severe malaria. A multiple linear regression analysis and simple bar was used to investigate associations between various haematological parameters.Malaria continues to be a global health challenge, affecting more than half the world’s population and causing approximately 660,000 deaths annually. The majority of malaria cases are caused by To examine the levels of erythropoietin in the age categories, the levels of erythropoietin was measured using a commercial Enyme-Linked Immunosorbent Assay (ELISA). Giemsa-stained blood smears were used to determine percentage pigment containing monocytes. The haemozoin containing monocytes was expressed as a percentage of the total number of monocytes. To obtain the number of haemozoin containing monocytes/μL the percentage of haemozoin containing monocytes was multiplied by the absolute number of monocytes/μL from the automated haematology analyzer.The levels of erythropoietin in younger children (<3 years) was significantly higher than in older children with a similar degree of malaria anaemia (Hb levels) (p < 0.005). Haemozoin-containing monocytes were relatively higher in severe malaria anaemia patients compared to those with uncomplicated malaria (p < 0.001).Age purportedly has a direct effect on background levels of erythropoietin. With corresponding decreased levels of erythropoietin in older children with the same degree of severe malarial anaemia, conceivably, the bone marrows of younger children with acute malaria may be less sensitive to erythropoietin. Malaria is an infectious disease and a major public health problem. Malaria can be uncomplicated or severe, often manifesting as severe anaemia, impaired consciousness or respiratory distress . MetabolP. falciparum asexual parasitemia, in the absence of any other or additional identifiable cause of anaemia [Plasmodium falciparum infection, especially among children . The anaemia of malaria has profound pathological effects on patients and its root cause is multifactorial and encompasses haemolysis, erythrophagocytosis, dyserythropoiesis, and ineffective erythropoiesis [Malaria contributes significantly to the development of anaemia in sub-Saharan Africa and elsewhere, where the disease is prevalent . Indeed  anaemia , is the opoiesis –6. Its mIn addition to direct destruction of infected erythrocytes by the malaria parasites during schizogony and the premature removal of non-parasitized erythrocytes, reversible suppression of erythropoiesis has also been reported to play a key role in its pathogenesis . SuppresThis study was hospital-based,, involving children aged 1–13 years, admitted to the emergency unit of the Department of Child Health (DCH) of the Korle-Bu Teaching Hospital and outpatients at the Korle-Bu Polyclinic, both in Accra, Ghana during the peak malaria transmission season . The patients were examined clinically by a physician and laboratory investigations including haematology, microscopy and clinical chemistry carried out.Parents/guardians of patients gave informed consent prior to recruitment. Demographical, clinical history and management/outcomes were also documented. This study formed part of a larger one, which was approved by the Institutional Review Board (IRB) and the Scientific and Technical Committee (STC) of the Noguchi Memorial Institute for Medical Research (NMIMR), College of Health Sciences, University of Ghana, Legon. The study also received the approval of the Scientific and Ethical Review Committee of the University of Ghana Medical School, College of Health Sciences, and University of Ghana.Inclusion criteria for malaria patients were fever ≥37.5°C) measured within 24 hours of admission, malaria parasitaemia, and at least one other sign of malaria . Children with other clinical presentations other than malaria or that had a positive sickling test were excluded from the study. Patients were categorized as uncomplicated and severe malaria based on previously established clinical characterization, haematological and other indices 7.5°C mea.Plasmodium falciparum parasitaemia, were fully conscious without severe bleeding or convulsions and had no other cause of anaemia. SMA also included patients who were unconscious with a Blantyre coma score of ≤3, duration of coma >60 minutes, any Hb value, any parasitaemia and no other neurological diseases [Severe malaria cases had haemoglobin (Hb) ≤5g/dL and any level of peripheral blood Venous blood was collected into sterile vacutainer tubes containing heparin and EDTA for routine diagnostic purposes. Plasma was obtained by centrifugation using the poor-platelet plasma (PPP) method. In this method, the vacutainer tube containing blood samples was centrifuged at 65 × g for 15 minutes at room temperature. The supernatant which is the Rich-Platelet-Protein (RPP) fraction was taken without disturbing the buffy coat and put into another tube. This was again centrifuged at 960 × g for 10 minutes. The supernatant which is the PPP was then collected and stored at −80 degrees Celsius.Thick and thin blood films obtained at enrollment prior to therapy were stained with Giemsa for detection, identification and enumeration of Plasmodium. Peripheral parasite density was determined from thick films based on the number of asexual forms against 300 white blood cells (WBCs) and the value multiplied by each individual’s WBC count from the automatic haematology analyzer  to obtain parasites per μL of blood. Absolute parasite counts were calculated for each child using either thin films (counting parasitized RBCs per 500 RBCs), or from thick films (counting parasitized RBCs per 300 WBCs).The Quantikine IVD Epo ELISA kit was used to determine erythropoietin levels following the manufacturer’s instructions.http://www.sigmaplot.com) and graphpad prism 5. Spearman’s rank coefficients were calculated to assess correlations between various parameters and levels of haemoglobin as well as levels of haemozoin containing monocytes and neutrophils.Statistical analyses were carried out using Sigmaplot 12  . This fiPrevious studies have established the ability of erythropoietin to cross the blood/brain barrier and to provide protection against experimental injury as well as act as a tissue protective cytokine , 14. It In the present study percentage haemozoin containing monocytes were significantly elevated in severe malaria anaemia compared to uncomplicated malaria. This finding corroborates earlier findings which implicated haemozoin in malaria pathogenesis through the suppression of erythropoiesis directly or in synergy with proinflammatory cytokines . The eleIn conclusion this study has confirmed earlier findings that erythropoietin production is age-related with younger children having the ability to produce more erythropoietin during acute malaria than older children. This study also suggests the involvement of haemozoin a parasite product in the inhibition of erythropoiesis as the seeming robust production of erythropoietin did not translate into higher Hb levels.


Plasmodium vivax Merozoite Surface Protein-1 (MSP-1) is an antigen considered to be one of the leading malaria vaccine candidates. PvMSP-1 is immunogenic and evidences suggest that it is a target for protective immunity against asexual blood stages of malaria parasites. This study aims to evaluate the acquired cellular and antibody immune responses against PvMSP-1 in individuals exposed to malaria infections in a Brazilian malaria endemic area.19) and P. falciparum PSS1 crude antigen. Antibodies IgE, IgM, IgG and IgG subclass and the levels of TNF-α, IFN-γ and IL-10 were measured by enzyme-linked immunosorbent assay (ELISA).The study was carried out in Paragominas, Pará State, in the Brazilian Amazon. Blood samples were collected from 35 individuals with uncomplicated malaria. Peripheral blood mononuclear cells were isolated and the cellular proliferation and activation was analyzed in presence of 19 kDa fragment of MSP-1 (PvMSP-1+was greater than CD8+ T cells, in both ex-vivo and in 96 h culture in presence of PvMSP-119 and PSS1 antigen. A low proliferative response against PvMSP-119 and PSS1 crude antigen after 96 h culture was observed. High plasmatic levels of IFN-g and IL-10 as well as lower TNF levels were also detected in malaria patients. However, in the 96 h supernatant culture, the dynamics of cytokine responses differed from those depicted on plasma assays; in presence of PvMSP-119 stimulus, higher levels of TNF were noted in supernatant 96 h culture of malaria patients’ cells while low levels of IFN-g and IL-10 were verified. High frequency of malaria patients presenting antibodies against PvMSP-119 was evidenced, regardless of class or IgG subclass. PvMSP-119-induced antibodies were predominantly on non-cytophilic subclasses.The prevalence of activated CD419 was able to induce a high cellular activation, leading to production of TNF and emphasizes the high immunogenicity of PvMSP-119 in naturally exposed individuals and therefore its potential as a malaria vaccine candidate.PvMSP-1


The pathogenesis of malaria during pregnancy is not completely understood. There are few published data on complement activation and malaria during pregnancy. This study aimed to investigate complement activation and malaria during pregnancy, and their association with hemoglobin and birth weight.A cross-sectional study was conducted at Medani, Sudan. Soluble terminal complement complex (TCC) levels were measured using ELISA in maternal and cord blood samples from 126 parturient women.Plasmodium falciparum-positive blood films from maternal peripheral blood, the placenta, or cord blood samples. Three (2.4%) and 22 (17.5%) of the placentas showed chronic and previous infection with histopathological examination, respectively, while 101 (80.2%) of them had no malaria infection. The mean [SD] of the maternal  and cord blood  TCC levels were significantly lower in cases of placental malaria infection (n = 25) than in those without placental malaria infection (n = 101). Linear regression showed that placental malaria infection was significantly associated with birth weight , but there were no associations between maternal and cord TCC levels and maternal hemoglobin, or between TCC levels and birth weight.There were no Maternal and cord blood TCC levels are lower in women with placental malaria infection than in those without placental malaria infection.http://www.diagnosticpathology.diagnomx.eu/vs/9600054761463915The virtual slide(s) for this article can be found here:  Malaria is a large public health problem in endemic tropical countries, especially sub-Saharan Africa. In sub-Saharan Africa, approximately 125 million pregnant women live in malaria-endemic areas and 32 million of these pregnant women are at risk of malaria -8.Plasmodium falciparum placental malaria infection compared with the malaria-negative group [The exact mechanism by which malaria infection and placental inflammation result in fetal growth restriction and LBW is poorly understood. However, many chemokines and inflammatory cytokines are associated with malaria infection and malaria-related LBW . The comve group . FurtherThe current study was conducted in Medani Maternity Hospital, Central Sudan, to investigate TCC levels in women with placental malaria, and their effect on maternal hemoglobin and birth weight.P. falciparum is the main malaria parasite species in the area and transmission occurs during the rainy (July to September) and post-rainy season [A cross-sectional study was conducted during August to December 2011 (the rainy and post-rainy season) in the labor ward of the Medani Maternity Hospital. The area of this study is characterized by unstable malaria transmission. y season . Medani The total sample size was calculated to have over 80% power to detect a difference of 5% at α = 0.05. We assumed that 10% of women might not respond or have incomplete data.2. Newborns were weighed immediately following birth using the Salter scale and the sex of each newborn was recorded.After obtaining signed informed consent from the patients, information on socio-demographics, history of obstetrics, medical history, antennal attendance characteristics, and bed net use was gathered using structured questionnaires. Body mass index was calculated by measuring maternal weight and height, which was expressed as weight (kg)/height (m)Maternal, placental, and cord blood films were prepared. Slides were stained by 10% Giemsa and the number of asexual parasites was counted per 200 leukocytes, assuming a leukocyte count of 8000 leukocytes/μl (for thick films) or per 1000 red blood cells (for thin films). Blood films were considered negative if no parasites were detected in 100 oil immersion fields of a thick blood film, which was double-checked in a blind manner by an expert microscopist. Maternal hemoglobin concentrations were estimated by the HemoCue hemoglobinometer .The blood  was then allowed to clot and centrifuged for 10 minutes at 3000 rpm and the serum was separated and stored at - 20°C till the analyses.3 sample was obtained from the maternal surface approximately half the distance between the umbilical cord and the edge of the placenta. Each biopsy sample was immediately placed in 10% neutral buffered formalin. Buffer was used to prevent formation of formalin pigment, which has similar optical characteristics and polarized light activity as malaria pigment [The details of how placental histology was performed have been mentioned previously ,18,19. B pigment . All of  pigment : uninfecMaternal and cord serum levels were measured using a human TCC ELISA kit  by following the manufacturer’s protocol.t test. Multivariate analyses were performed using binary models for placental malaria infection as the dependent variable and linear models with hemoglobin, birth weight, and TCC  levels as continuous dependent variables. Socio-demographic characteristics, education, antenatal care, residence, and placental malaria infections were the independent predictor of interest. Odds ratios (OR) and 95% confidence intervals (CI) were calculated and a P value of <0.05 was considered significant.Data were entered into a computer using SPSS for windows (version 16.0). Continuous data (including TCC levels) were normally distributed and were compared between groups using Student’s The study received ethical clearance from the Research Board at the Faculty of Medicine, University of Khartoum, Sudan.Out of the 126 women enrolled in the study, 51 (41.5%) were primiparae and 62 (49.2%) had rural residency. Two (1.6%), 33 (26.2%), and 91 (92.1%) of these 126 women had no, one to two visits, and more than two visits of antenatal care, respectively. The majority  of them used bed nets during the index pregnancy.Thirty one (24.6%) of these 126 women had blood group A, 17 (13.5%) had blood group B, three (2.4%) had blood group AB, and 75 (59.5%) had blood group O. The mean (SD) hemoglobin level was 10.7 (1.1) g/dl, and 72 (57.1%) of the women were anemic (hemoglobin <11 g/dl).Eleven (8.7%) women delivered low-birth weight neonates (<2500 g).P. falciparum-positive blood films from maternal peripheral blood, the placenta, or cord blood samples. Three (2.4%) and 22 (17.5%) of the placentas showed chronic and previous infection on a histopathological examination, respectively, while 101 (80.2%) of them showed no signs of malaria infection. There were no significant associations between age, parity, residence, antenatal care, blood group, and placental malaria infection (Table There were no ) and 22 7.5% of tBirth weight was significantly lower in women with placental malaria infection n = 25) compared women who did not have placental malaria infection . In linear regression, placental malaria infection was significantly associated with birth weight  and cord  blood TCC levels were significantly lower in women with placental malaria infection (n = 25) than in those without placental malaria infection  and cord blood TCC levels  in infected and non-infected women, even when the malaria-infected women were analyzed separately .The main findings of the current study were that there were no associations between malaria placental infection and age or parity. Birth weight was significantly lower in newborns of malaria-infected women than in non-infected women. Furthermore, maternal and cord blood TCC levels were significantly lower in women with placental malaria infection than in those without placental malaria infection, and they had no effect on maternal hemoglobin levels and birth weight. The susceptibility of pregnant women to malaria infection, irrespective of their age or parity, is similar to previous findings in the same setting, as well as in different areas of Sudan ,19,22,23et al. observed that cytokine levels  were significantly lower in peripheral and placental sera of women with placental malaria infection in eastern Sudan [et al. reported significantly higher levels of TCC in women with P. falciparum placental malaria infection compared with the malaria-negative group [In the current study, maternal and cord blood TCC levels were significantly lower in women with placental malaria infection than in those without placental malaria infection. Bayoumi rn Sudan . Recentlrn Sudan ,14. Likeve group . NotablyP. falciparum malaria by the mechanism of reduced resetting [Interestingly, in this study, maternal and cord blood TCC levels were significantly lower in women with an O blood group compared with those with other blood types. Although there was no association between the O blood group and placental malaria infection in the current study, a previous study in eastern Sudan showed that women with blood type O were at higher risk of placental malaria infection  . Howeveresetting .In the current study, although placental malaria infection was associated with maternal and cord blood TCC levels and birth weight, there was no association between either maternal or cord blood TCC levels and birth weight. Recent observations have shown that TCC levels are higher in placental plasma samples of newborns weighing less than 2700 g than in heavier newborns . SubmicrThere are significantly lower levels of maternal and cord blood TCC levels in women with placental malaria infection than in those without placental malaria infection. There is no association between maternal hemoglobin and TCC  levels or between TCC levels and birth weight.


Plasmodium falciparum have become a vital tool to accelerate vaccine and drug development. As CHMI trials are carried out in a controlled environment, they allow unprecedented, detailed evaluation of parasite growth dynamics and immunological responses to infection. Though commonly performed in malaria-naïve populations, CHMI trials have rarely been performed in malaria endemic regions, despite the advantages of early testing of candidate anti-malaria vaccines and drugs in populations where malaria is endemic. To date, modern CHMI studies have not been used to investigate mechanisms of naturally acquired immunity (NAI) to P. falciparum infection.Controlled human malaria infection (CHMI) studies, where healthy volunteers are infected with P. falciparum sporozoites  administered intramuscularly to evaluate safety, infectivity and parasite growth dynamics in healthy Kenyan adults (n = 28). We included adults with varying degrees of prior exposure to malaria in order to allow an evaluation of the effect of NAI on parasite growth dynamics post CHMI.We conducted an open label, randomized CHMI pilot study using aseptic, cryopreserved P = 0.044).All participants developed blood-stage infection, with a similar safety profile to that observed in northern CHMI centres. One volunteer remained asymptomatic and blood-film negative until day 21 post injection of PfSPZ Challenge, despite developing confirmed blood-stage infection by quantitative polymerase chain reaction (qPCR). This volunteer had a notably reduced parasite multiplication rate (1.3) in comparison to the other 27 volunteers (median 11.1). A significant correlation was seen between parasite multiplication rate (PMR) and anti-schizont ELISA OD at screening (P. falciparum malaria.Our study has shown that the PfSPZ Challenge is safe and infectious in malaria endemic populations and could be used to aid early assessment of the efficacy of candidate malaria vaccines and drugs in African populations. Whilst our findings are limited by sample size, our pilot study proves that NAI can impact on PMR post-CHMI in a detectable fashion. Our findings suggest the CHMI sporozoite model could allow identification of a heterogeneous group of participants with varying degrees of NAI for whom the relationship between PMR and candidate assays of blood-stage immunity can be examined to try to gain greater understanding of the mechanisms of NAI to


Current artesunate (ARS) regimens for severe malaria are complex. Once daily intramuscular (i.m.) injection for 3 d would be simpler and more appropriate for remote health facilities than the current WHO-recommended regimen of five intravenous (i.v.) or i.m. injections over 4 d. We compared both a three-dose i.m. and a three-dose i.v. parenteral ARS regimen with the standard five-dose regimen using a non-inferiority design (with non-inferiority margins of 10%).n = 348) or three injections of 4 mg/kg  either i.m. (n = 348) or i.v. (n = 351), both of which were the intervention arms. The primary endpoint was the proportion of children with ≥99% reduction in parasitemia at 24 h from admission values, measured by microscopists who were blinded to the group allocations. Primary analysis was performed on the per-protocol population, which was 96% of the intention-to-treat population. Secondary analyses included an analysis of host and parasite genotypes as risks for prolongation of parasite clearance kinetics, measured every 6 h, and a Kaplan–Meier analysis to compare parasite clearance kinetics between treatment groups. A post hoc analysis was performed for delayed anemia, defined as hemoglobin ≤ 7g/dl 7 d or more after admission.This randomized controlled trial included children (0.5–10 y) with severe malaria at seven sites in five African countries to assess whether the efficacy of simplified three-dose regimens is non-inferior to a five-dose regimen. We randomly allocated 1,047 children to receive a total dose of 12 mg/kg ARS as either a control regimen of five i.m. injections of 2.4 mg/kg  ; 139 participants were lost to follow-up. In the three-dose i.m. arm, 265/338 (78%) children had a ≥99% reduction in parasitemia at 24 h compared to 263/331 (79%) receiving the five-dose i.m. regimen, showing non-inferiority of the simplified three-dose regimen to the conventional five-dose regimen . In the three-dose i.v. arm, 246/333 (74%) children had ≥99% reduction in parasitemia at 24 h; hence, non-inferiority of this regimen to the five-dose control regimen was not shown . Delayed parasite clearance was associated with the N86YPfmdr1 genotype. In a post hoc analysis, 192/885 (22%) children developed delayed anemia, an adverse event associated with increased leukocyte counts. There was no observed difference in delayed anemia between treatment arms.The per-protocol population was 1,002 children  every year.Globally, about 200 million cases of malaria—a mosquito-borne parasitic disease—occur every year. Malaria infections, which can be caused by several parasites, can be “uncomplicated” or “severe.” Prompt treatment of uncomplicated malaria, which presents with flu-like symptoms, is essential to prevent the development of severe malaria. The World Health Organization (WHO) recommends artemisinin combination therapy (ACT) for the first-line treatment of uncomplicated malaria in countries where the disease is always present. In ACT, artemisinin derivatives  are combined with a slower-acting, more slowly eliminated partner drug to prevent the original infection recurring and to reduce the risk of the malaria parasites becoming resistant to either drug. Severe malaria, which is usually caused by WHO recommends that severe malaria be treated with intravenous or intramuscular injections of artesunate, a parenteral (injectable) form of artemisinin; patients with severe malaria cannot take pills reliably or safely. Specifically, WHO recommends that patients be given 2.4 mg of artesunate per kilogram of body weight intravenously or intramuscularly at the time of admission (0 hours) and at 12, 24, 48, and 72 hours (followed by ACT to ensure full parasite clearance). But this five-dose regimen is complex. A simpler regimen would be easier to administer in resource-limited settings, where giving the correct doses on time to small, sick children can be challenging. In this open-label, non-inferiority randomized controlled trial (RCT), the researchers investigate the efficacy of a three-dose artesunate regimen for the treatment of severe malaria in African children. RCTs compare outcomes in people randomly chosen to receive different interventions; in an open-label RCT, both the researchers and the participants know which treatment is being given; a non-inferiority trial investigates whether one treatment is not worse than another treatment.The researchers randomly allocated 1,047 children aged six months to ten years with severe malaria attending seven clinical centers in five African countries to receive a total dose of 12 mg of artesunate per kilogram of body weight as five intramuscular injections of 2.4 mg/kg given at 0, 12, 24, 48, and 72 hours (the control regimen) or as three intramuscular or intravenous injections of 4 mg/kg given at 0, 12, and 24 hours . The trial’s primary endpoint was the proportion of children whose parasitemia (parasite count in the blood) at 24 hours was ≤1% of that at admission . Among the 1,002 children who received the planned drug doses (the per-protocol population), 78% in the three-dose intramuscular group had ≥99% parasite clearance compared to 79% in the five-dose intramuscular group, a result that met a preset criterion for non-inferiority at 24 hours of the three-dose intramuscular regimen to the control regimen. However, because only 74% of the children in the three-dose intravenous group had ≥99% parasite clearance, this regimen was not shown to be non-inferior to the conventional five-dose regimen.These findings when combined with the findings of several secondary analyses suggest that, in African children, a three-dose intramuscular artesunate regimen is non-inferior to the WHO-recommended regimen for the treatment of severe malaria. The study’s open-label design may limit the accuracy of its findings, as may its use of a primary endpoint midway through drug treatment rather than at the end  and its use of parasite clearance rather than death as the primary endpoint . Overall, these findings support the use of the three-dose intramuscular artesunate regimen for the treatment of severe malaria. Importantly, however, 22% of the children in the study developed delayed anemia, irrespective of treatment regimen. Thus, although further studies are needed to clarify whether treatment with artesunate or the malaria infection itself was responsible for the delayed anemia, patients treated with artesunate for severe malaria should be routinely monitored for this complication.http://dx.doi.org/10.1371/journal.pmed.1001938.This list of resources contains links that can be accessed when viewing the PDF on a device or via the online version of the article at malaria ; the World Malaria Report 2014 provides details of the current global malaria situation, including information on malaria in individual African countries; WHO’s Guidelines for the Treatment of Malaria and its Management of Severe Malaria: A Practical Handbook are availableInformation is available from the World Health Organization on malaria (in English and Spanish), including personal stories about malariaThe US Centers for Disease Control and Prevention provides information on malaria, including a personal storyThe UK National Health Service Choices website also provides information about global control of malariaInformation is available from the Roll Back Malaria Partnership on the Scientists Against Malaria collaboration applies modern drug design and modeling techniques to develop new treatments against malaria; its website includes information about many aspects of malariaThe guidance and research and analysis on malariaPublic Health England provides a collection of malaria (in English and Spanish)MedlinePlus provides links to additional information on this trial is availableMore information about  Studies to optimize artesunate (ARS) treatment regimens in malaria have been surprisingly sparse, given that ARS is now established as the treatment of choice for severe malaria in both adults and children ,2. WHO rIn an earlier study, a simplified three-dose ARS i.v. regimen was found to be non-inferior in pharmacodynamic efficacy to the conventional WHO regimen , and itspfmdr1 and kelch-13 with parasite clearance kinetics and, in post hoc analysis, the occurrence of delayed anemia.We examined i.m. ARS in severe malaria in seven hospitals of the Severe Malaria in African Children (SMAC) network ,8. We asOur primary study objective was to assess the non-inferiority of i.v. ARS and i.m. ARS simplified dosing regimens  to the standard i.m. treatment dosing regimen  in clearing parasitemia in African children with severe malaria. Our secondary study objectives were to compare the tolerability and safety of the three ARS dosing regimens, to analyze host and parasite genotypes as risks for prolongation of parasite clearance kinetics, measured every 6 h, and to compare parasite clearance kinetics between treatment groups. An exploratory objective was to analyze genetic polymorphisms in humans and parasites linked to disease and treatment, and a post hoc objective was to assess the occurrence of delayed anemia.The trial was performed according to the principles of the Declaration of Helsinki and Good Clinical Practice. Ethics committees and competent authorities for each study site approved the study. A data monitoring board (DMB) provided oversight for the study with respect to safety and efficacy as well as appropriate implementation of the defined stopping rules.Plasmodium falciparum malaria. Patients  or two (control arm) doses of ARS had been administered. This endpoint was derived from discussions in the SMAC network and from our own studies . In seveEvaluation of the whole regimens is included in the secondary outcome measures (and there is no discrepancy between the results for this and the 24-h end point). Further endpoints were time to total clearance of asexual parasites, time to 99% reduction of asexual parasites, time to 90% reduction of asexual parasites, time to 50% reduction of asexual parasites, proportion of patients with genotype-uncorrected adequate clinical and parasitological response on day 28, percent reduction in asexual parasites from baseline at 24 h after initiation of randomized study drug, and percent reduction in asexual parasites from baseline at 48 h after initiation of randomized study drug.During the conduct of this study, several patients in the case series developed delayed hemolytic anemia following ARS therapy, mostly in the second and third week from the start of therapy –15. MotiP. falciparum infection  and clinical signs and symptoms severe enough to require hospitalization, according to the SMAC definition of severe malaria that best reflects the policies of African hospitals ). HbAC or HbAS genotype was not associated with delayed anemia, and there was no relationship between delayed anemia and PK parameters.G6PDH-deficient participants (A−) had more anemia on admission  in our patients may reflect the large artemisinin  component of the treatment course and efficacy of the combination partner (lumefantrine).The frequently observed  Senegal  and decr Senegal , with no Senegal . Our res50 of DHAG = 5.7 μM, mean of two experiments), DHAG plasma concentrations commonly peaked above 5 μM Click here for additional data file.S1 Table(DOCX)Click here for additional data file.S2 Table(DOCX)Click here for additional data file.S3 Table(DOCX)Click here for additional data file.S4 Table(DOCX)Click here for additional data file.S5 Table(DOCX)Click here for additional data file.S6 Table(DOCX)Click here for additional data file.S7 Table(DOCX)Click here for additional data file.S1 Text(PDF)Click here for additional data file.S2 Text(DOC)Click here for additional data file.