Plasmodium species, correct diagnosis is crucial for appropriate treatment and surveillance. Species misidentification by microscopy has been reported in areas co-endemic for vivax and falciparum malaria, and may be more frequent in regions where Plasmodium knowlesi also commonly occurs.In areas co-endemic for multiple Plasmodium falciparum, P. knowlesi, and Plasmodium vivax malaria.This prospective study in Sabah, Malaysia, evaluated the accuracy of routine district and referral hospital-based microscopy, and microscopy performed by an experienced research microscopist, for the diagnosis of PCR-confirmed Plasmodium infection were enrolled, including 130 with P. knowlesi, 122 with P. falciparum, 43 with P. vivax, one with Plasmodium malariae and eight with mixed species infections. Among patients with P. knowlesi mono-infection, routine and cross-check microscopy both identified 94 (72%) patients as “P. malariae/P. knowlesi”; 17 (13%) and 28 (22%) respectively were identified as P. falciparum, and 13 (10%) and two (1.5%) as P. vivax. Among patients with PCR-confirmed P. falciparum, routine and cross-check microscopy identified 110/122 (90%) and 112/118 (95%) patients respectively as P. falciparum, and 8/122 (6.6%) and 5/118 (4.2%) as “P. malariae/P. knowlesi”. Among those with P. vivax, 23/43 (53%) and 34/40 (85%) were correctly diagnosed by routine and cross-check microscopy respectively, while 13/43 (30%) and 3/40 (7.5%) patients were diagnosed as “P. malariae/P. knowlesi”. Four of 13 patients with PCR-confirmed P. vivax and misdiagnosed by routine microscopy as “P. malariae/P. knowlesi” were subsequently re-admitted with P. vivax malaria.A total of 304 patients with PCR-confirmed P. falciparum, P. vivax and P. knowlesi in a region where all three species frequently occur. Misdiagnosis of P. knowlesi as both P. vivax and P. falciparum, and vice versa, is common, potentially leading to inappropriate treatment, including chloroquine therapy for P. falciparum and a lack of anti-relapse therapy for P. vivax. The limitations of microscopy in P. knowlesi-endemic areas supports the use of unified blood-stage treatment strategies for all Plasmodium species, the development of accurate rapid diagnostic tests suitable for all species, and the use of PCR-confirmation for accurate surveillance.Microscopy does not reliably distinguish between  Plasmodium falciparum, the relative contribution of the non-falciparum Plasmodium species to the global malaria burden is increasing as incidence of P. falciparum falls [Plasmodium vivax, which accounts for half of the world’s malaria and is increasingly recognized as a cause of severe and potentially fatal disease [P. vivax has proved more difficult than P. falciparum, with successful malaria control programmes in some countries leading to an increase in incidence of P. vivax as overall malaria rates drop [Plasmodium knowlesi has been identified as the most common cause of human malaria in parts of Malaysia [Plasmodium species [Plasmodium knowlesi is capable of causing severe disease and death [Despite recent progress towards elimination, malaria continues to affect over 200 million people per year with an estimated 655,000 deaths . Althougum falls . The mos disease . Moreovetes drop . More reMalaysia -14, with species . Plasmodnd death ,13,15-18nd death .P. falciparum, P. vivax and P. knowlesi, species-differentiation at the time of diagnosis is crucial for directing appropriate treatment, particularly in settings which have separate treatment policies for different species, most commonly artemisinin-combination treatment (ACT) for P. falciparum and chloroquine for non-falciparum species. Even in regions such as Papua, Indonesia, which have adopted a unified treatment strategy of ACT for all malaria [P. vivax is still required to allow administration of anti-hypnozoite treatment to prevent relapses, with misdiagnosis of this species potentially leading to increased morbidity and transmission. In areas also endemic for P. knowlesi, accurate diagnosis is important for epidemiological surveillance of this potentially fatal emerging zoonotic infection.In areas co-endemic for  malaria , diagnosP. falciparum and P. vivax[P. knowlesi from Plasmodium malariae[P. knowlesi with other species may also be frequent [P. falciparum, P. knowlesi, and P. vivax, in an area where all three species commonly occur.Microscopy of stained blood smears remains the standard method of malaria diagnosis in most parts of the malaria-endemic world, and ideally allows differentiation of species. However, with the difficulty in distinguishing young ring-stage parasites, frequent misdiagnosis has been reported in areas co-endemic for  P. vivax,21. It i malariae,23, but frequent . This stThe study was conducted at Queen Elizabeth Hospital (QEH), an adult tertiary referral hospital in Kota Kinabalu, Sabah, Malaysia. The hospital services the West Coast and Kudat Divisions of Sabah, with six district hospitals and a population of 1.14 million. From September 2010, in response to ongoing malaria deaths in Sabah , new treAll patients referred to or admitted directly to QEH with a microscopic diagnosis of malaria were assessed for eligibility from September 2010 to October 2011 as part of a prospective study of the epidemiology, clinical spectrum and pathophysiology of knowlesi malaria, reported elsewhere . Non-preP. knowlesi from P. malariae is not possible [P. malariae, P. knowlesi, or P. malariae/P. knowlesi were all considered a single group, further referred to as “P. malariae/P. knowlesi”. Parasite density was quantified by the research microscopist using pre-treatment slides, and reported as the number of parasites per 200 white blood cells or per 1,000 red blood cells and converted to parasites/μL using the patient’s white blood cell count or haematocrit, respectively. If a pre-treatment slide could not be reliably obtained  the referring hospital’s microscopy report was used and the “1+ − 4+” grade converted into parasites/μL using the relevant median parasite density. Parasite DNA was extracted and PCR performed using previously described methods for P. falciparum, P. vivax, Plasmodium ovale, and P. malariae[P. knowlesi[Standardized data forms were used to record demographic and clinical information. Venous blood was collected in a CTAD tube labelled with the patient’s study number, and thick and thin blood smears prepared using Giemsa staining. Species identification using thick and thin blood films was performed initially by microscopists at referring district hospitals, or at QEH if presenting directly to this hospital (routine microscopy). Thick and thin films were later cross-checked by a research microscopist (cross-check microscopy) with more than 15 years’ experience, who was blinded to the results of routine microscopy. Because reliable differentiation of possible , slides  malariae and P. k knowlesi. PCR dia2 test was used for intergroup differences between categorical variables. Logistic regression was used to assess the association between parasite count and correct identification of species.Data were analysed using STATA version 10.1 . For continuous variables intergroup differences were compared using the Kruskal-Wallis test, or the Mann–Whitney test for pairwise comparisons, while the χPlasmodium infection were enrolled, including 130 with P. knowlesi, 122 with P. falciparum, 43 with P. vivax, one with P. malariae, and eight with mixed-species infection. Demographic and clinical features are reported separately [P. falciparum, and 21 (49%) with P. vivax malaria. Severe malaria occurred in 38 (29%) patients with P. knowlesi, 13 (11%) with P. falciparum, and seven (16%) with P. vivax[P. knowlesi had lower parasite counts than those with non-severe P. falciparum , but not P. vivax . Among patients with severe malaria, there was no significant difference in median parasitaemia of those with P. knowlesi  and P. falciparum .A total of 304 patients with PCR-confirmed parately . Half  patients as “P. malariae/P. knowlesi”, with 66 (51%) patients correctly diagnosed by both microscopy readings. Routine microscopy diagnosed 17 (13%) and 13 (10%) patients with PCR-confirmed P. knowlesi mono-infection as P. falciparum and P. vivax respectively, while cross-check microscopy diagnosed 28 (22%) and two (1.5%) as P. falciparum and P. vivax respectively. Four (3%) patients with PCR-confirmed P. knowlesi were diagnosed by both routine and cross-check microscopy as P. falciparum, and no patient was diagnosed by both readings as P. vivax. Among the 38 patients with severe knowlesi malaria, routine microscopy correctly diagnosed 33 (87%) as “P. malariae/P. knowlesi”. Three were diagnosed as P. falciparum, one as P. vivax and one as P. falciparum/“P. malariae/P. knowlesi”. The median parasite count among the five patients with severe knowlesi malaria and misdiagnosed by routine microscopy was 168,279  parasites/μL, and was not significantly different to those correctly diagnosed.Microscopy and PCR results are shown in Tables P. falciparum, routine and cross-check microscopy correctly identified 110/122 (90%) and 112/118 (95%) as P. falciparum, while 8/122 (6.6%) and 5/118 (4.2%) patients respectively were diagnosed as “P. malariae/P. knowlesi”. All 13 patients with severe falciparum malaria were correctly diagnosed by routine microscopy.Among patients with PCR-confirmed P. vivax, 23/43 (53%) and 34/40 (85%) were correctly diagnosed by routine and cross-check microscopy respectively, while 13/43 (30%) and 3/40 (7.5%) patients were diagnosed as “P. malariae/P. knowlesi”. Among the 13 patients with PCR-confirmed P. vivax and misdiagnosed by routine microscopy as “P. malariae/P. knowlesi”, four (31%) were re-admitted to QEH within four months with PCR-confirmed P. vivax infection. Among the seven patients with severe vivax malaria, routine microscopy diagnosed three as P. vivax, two as “P. malariae/P. knowlesi”, one as P. falciparum/P. vivax, and one as “P. malariae/P. knowlesi”/P. vivax.Among those with PCR-confirmed P. falciparum/P. vivax was correctly identified by routine microscopy, and another with P. falciparum/P. vivax was correctly identified by cross-check microscopy. All others were misdiagnosed by both microscopic readings (with one patient’s slide unavailable for cross-check microscopy).Among the eight patients with mixed-species infection by PCR, one patient with P. vivax by routine microscopy , with a similar trend also seen with identification of P. vivax by cross-check microscopy . No association however occurred between parasite count and correct identification of P. knowlesi or P. falciparum, by either routine or cross-check microscopy.An association was found between parasite count and correct identification of Plasmodium species in an area where P. falciparum, P. vivax and P. knowlesi all commonly occur. Misdiagnosis of P. knowlesi as both P. vivax and P. falciparum, and vice versa, is common. Only 72% of patients with PCR-confirmed P. knowlesi received an accurate diagnosis by routine or by cross-check microscopy, and correlation between the microscopic methods was poor, with even fewer patients receiving an accurate diagnosis of P. knowlesi by both methods. These findings occurred despite considering P. knowlesi and P. malariae as a single group, and so were not a consequence of the well described near impossibility of distinguishing these two species. Rather, patients with PCR-confirmed P. knowlesi were commonly misdiagnosed as having either P. falciparum or P. vivax malaria, with misdiagnosis of P. vivax as “P. malariae/P. knowlesi” also common.This study highlights the difficulties of microscopic diagnosis of P. knowlesi from P. falciparum by microscopy has been previously described, due to similarities between the young rings of P. knowlesi and ring forms of P. falciparum, including double chromatin dots, multiple-infected erythrocytes and applique forms [P. malariae” were actually P. falciparum by PCR, and 33/312 (11%) microscopy-diagnosed P. falciparum cases were P. knowlesi by PCR [P. knowlesi and P. vivax was also notable. In the current study 30% of patients with PCR-confirmed P. vivax were misdiagnosed by routine microscopy as “P. malariae/P. knowlesi”, with four patients subsequently re-admitted with presumed vivax relapses due to lack of primaquine treatment. In the Sarawak study, 43 of 440 (10%) patients with PCR-confirmed P. vivax malaria were misdiagnosed as “P. malariae” [P. knowlesi was misdiagnosed as P. vivax by microscopy and a fatal case of P. vivax was misdiagnosed as “P. malariae” [The difficulty with distinguishing ue forms ,23. In ai by PCR . In thisalariae” . In a sealariae” .P. vivax as “P. malariae/P. knowlesi” has significant implications for malaria control, as failure to administer anti-hypnozoite treatment may lead to increased transmission and may hamper efforts to eliminate vivax malaria in regions where P. knowlesi is common. These findings support the current Sabah Ministry of Health policy for the performance of reference centre PCR on all patients with a microscopic diagnosis of “P. malariae/P. knowlesi” to enable administration of primaquine to those found to have misdiagnosed P. vivax. In knowlesi-endemic areas, where logistically possible, PCR should also be performed on at least a proportion of slides diagnosed as P. falciparum or P. vivax, to allow monitoring of the accuracy of microscopic diagnoses at different clinical sites, and to identify areas where additional training of microscopists may be required. Given the inaccuracies of microscopic diagnosis, performance of PCR is also essential to maintain accurate surveillance, particularly monitoring the emergence of P. knowlesi.This frequent misdiagnosis of Plasmodium species creates difficulties with basing treatment decisions on microscopic results. The 2008 Malaysian Ministry of Health malaria treatment guidelines recommend chloroquine for uncomplicated P. malariae/P. knowlesi malaria; chloroquine plus primaquine for uncomplicated P. vivax malaria; artemisinin combination treatment  for uncomplicated P. falciparum malaria; and intravenous artesunate, or intravenous quinine plus oral doxycycline, for severe P. falciparum or severe P. malariae/P. knowlesi malaria [P. falciparum or P. malariae/P. knowlesi malaria; chloroquine plus primaquine, or ACT, for uncomplicated P. vivax malaria; and intravenous artesunate  for severe malaria from any species [The inaccuracy of microscopy for differentiating  malaria . No reco species .P. falciparum as P. knowlesi would, under current national guidelines, result in administration of chloroquine for P. falciparum. Given widespread resistance of P. falciparum to chloroquine, this would lead to increased risk of complications and/or fatal outcome, as well as increased transmission. Patients with severe P. knowlesi malaria misdiagnosed as P. vivax may fail to receive immediate parenteral treatment if national guidelines are followed, and this scenario has been previously associated with fatal outcomes [P. knowlesi as P. vivax may still lead to treatment with oral chloroquine if signs of severity are missed, potentially leading to adverse outcomes given the slower parasite clearance associated with chloroquine as compared to oral ACT [Given the different treatment recommendations for each species in both of these guidelines, inappropriate treatment decisions may be made as a result of incorrect microscopic diagnoses. Misdiagnosis of outcomes . Even iforal ACT .Plasmodium species in knowlesi-endemic areas, an approach increasingly recommended for regions co-endemic for P. falciparum and P. vivax[P.knowlesi remains undetermined.These results therefore support the argument for a unified treatment strategy of ACT for uncomplicated malaria from all  P. vivax. These d P. vivax. Even if P. vivax, althougP. falciparum misdiagnosed as another species. Furthermore, chloroquine-resistant P. vivax is an increasing problem throughout Southeast Asia [P. vivax malaria may therefore be associated with treatment failures, and may potentiate spread of chloroquine resistance.An additional advantage of this unified treatment approach would be the avoidance of inadvertent use of chloroquine for ast Asia , and hasast Asia  and Peniast Asia -35. Use Plasmodium species, in order that reliable results can be obtained more quickly and cheaply than is possible with PCR. Although histidine-rich protein 2 (HRP2)-based RDTs are able to diagnose P. falciparum, RDTs that distinguish P. knowlesi from P. vivax are not yet available. Limited data suggest that P. knowlesi cross-reacts with both P. falciparum and P. vivax-specific pLDH [P. vivax, P. falciparum and P. knowlesi mono-infections. However, while a pLDH RDT has shown high sensitivity for the diagnosis of severe malaria from all three of these species, neither pLDH- or aldolase-based RDTs have demonstrated sufficiently high sensitivity for uncomplicated P. knowlesi[Finally, this study highlights the need to develop rapid diagnostic tests (RDTs) that have the ability to distinguish between fic pLDH -39, and  knowlesi. ProspecP. falciparum, P. vivax and P. knowlesi in areas co-endemic for all three species, with misdiagnosis of P. vivax and P. knowlesi particularly common. In P. knowlesi-endemic areas these limitations of microscopic diagnosis must be considered when developing strategies to monitor the prevalence of the different malaria species, and when developing treatment guidelines.This study has found that microscopy does not reliably distinguish between The authors declare that they have no competing interests.NMA, TWY, TW and BEB conceived and designed the study; BEB collected and analysed the data. BEB and NMA wrote the manuscript. MJG assisted with data collection. All authors read and approved the final manuscript.


Plasmodium vivax infections associated with severe and fatal disease, the identification of sensitive and reliable markers for vivax severity is crucial to improve patient care. Circulating nucleic acids (CNAs) have been increasingly recognized as powerful diagnostic and prognostic tools for various inflammatory diseases and tumors as their plasma concentrations increase according to malignancy. Given the marked inflammatory status of P. vivax infection, we investigated here the usefulness of CNAs as biomarkers for malaria morbidity.Given the increasing evidence of P. vivax malaria patients from the Brazilian Amazon and 14 malaria non-exposed healthy donors were quantified by two different methodologies: amplification of the human telomerase reverse transcriptase (hTERT) genomic sequence by quantitative real time PCR (qPCR), and the fluorometric dsDNA quantification by Pico Green. CNAs levels were significantly increased in plasma from P. vivax patients as compared to healthy donors (p<0.0001). Importantly, plasma CNAs levels were strongly associated with vivax morbidity (p<0.0001), including a drop in platelet counts (p = 0.0021). These findings were further sustained when we assessed CNAS levels in plasma samples from 14 additional P. vivax patients of a different endemic area in Brazil, in which CNAS levels strongly correlated with thrombocytopenia (p = 0.0072). We further show that plasma CNAs levels decrease and reach physiological levels after antimalarial treatment. Although we found both host and parasite specific genomic sequences circulating in plasma, only host CNAs clearly reflected the clinical spectrum of P. vivax malaria.CNAs levels in plasma from twenty-one acute Here, we provide the first evidence of increased plasma CNAs levels in malaria patients and reveal their potential as sensitive biomarkers for vivax malaria morbidity. Plasmodium vivax malaria threatens almost 40% of the world's population, with an upper estimate of 300 million cases each year P. vivax infections with severe or even fatal outcomes, with incidence and morbidity rates similar to those for P. falciparum. Consequently, costs due to hospitalization have significantly raised as well as the need for intensive care, which helped vivax malaria to be placed in a higher status of public health emergency In the last decade, a series of case reports and longitudinal studies carried out in India P. vivax infections Compared to falciparum malaria, there are remarkably large knowledge gaps in the pathophysiology of vivax malaria, and the true spectrum of clinical disease in endemic areas remains unknown Most of the DNA and RNA in the human body are located within cells, but small physiologic amounts of nucleic acids can also be found circulating freely in the blood. These DNA, RNA, and small RNA molecules may arise from both: i) active release of nucleic acids from living cells, or ii) break down of dying cells that release their contents into the blood. The term Circulating Nucleic Acids (CNAs) refers to cell free segments of DNA or RNA found in the bloodstream. Their existence in human plasma was first reported more than 60 years ago P. vivax, herein we tested the usefulness of plasma CNAs levels as markers for the morbidity of vivax malaria. We investigated the CNAs levels in plasma from P. vivax infected patients with different clinical presentations and found significant higher levels of CNAs in P. vivax infected patients, as compared to age-matched healthy donors. We found that plasma CNAs levels were closely correlated with variations in body temperature, platelets counts, and increased in a linear fashion with the clinical spectrum of vivax malaria, evaluated here by scoring patients' clinical and hematological parameters.Aiming at finding sensitive and reliable biomarkers for P. vivax patients by qPCR amplification of the genomic sequence of the human single copy gene hTERT and by fluorometric quantification of the dsDNA content with the Quant-iT™ Pico Green Reagent. The amplification plot of hTERT shows that the mean cycle threshold (Ct) achieved in CNAs samples from P. vivax patients (mean Ct 28.6±1.5) was significantly lower than the one reached in samples from healthy donors (mean Ct 31.5±0.79) (p<0.0001)  and healthy donors (0.1098 pg/ml) was confirmed when a standard curve, built from a serial dilution of an amplified sample of hTERT sequence, was used to interpolate the hTERT concentrations in the samples .CNAs levels were measured in plasma from <0.0001) . As the  samples . To norm samples . As expeP. vivax patients were confirmed by quantification of dsDNA with Quant-iT™ Pico Green Reagent (p<0.0001).The increased levels of total CNAs in plasma from  Reagent   compared to plasma samples from non-febrile patients, as revealed by the two different methodologies: amplification of hTERT genomic sequence by qPCR (p = 0.0376) and the quantification of dsDNA content with Quant-iT™ Pico Green (p = 0.0023) (data not shown).To investigate the potential of CNAs as biomarkers for malaria morbidity, we compared the levels of CNAs in plasma from patients with different clinical presentations, and scored according to clinical and hematological parameters . Figure r = 0.4795, p = 0.0034) was found between the CNAs levels and the intensity of clinical malaria. These data were confirmed when the Cts from the amplification of hTERT were analyzed   .r = −0.6451, p = 0.0021)   . These f 0.0027) .P. vivax morbidity, we assessed the CNAS levels in plasma from an additional group of P. vivax patients whose selection was carried-out in a different hospital of the Amazon area, Cuiaba, MT . Once the clinical protocol used at the hospital in Cuiaba was different from Manaus (FMT-HVD), we were unable to build a similar clinical score. For this reason, we compared the CNAS levels in these samples with thrombocytopenia, a common hematological disturbance seen in malaria morbidity in the Amazon area p = 0.0072) between CNAS levels and thrombocytopenia in P. vivax patients from Cuiaba . CNAs levels were significantly higher in samples from falciparum malaria patients as compared to healthy donors . Importantly, CNAs levels in patient's plasma clearly correlated with thrombocytopenia . The comparison of the mean Ct obtained after qPCR amplification of the hTERT in plasma samples from acute vs. treated patients confirmed these findings (p = 0.0243) , the CNAs levels were further assessed 7 days after antimalarial chemotherapy. As shown in  0.0243) . Seven d 0.0243) . These d 0.0243) . In thosP. vivax derived-CNAs in plasma in an attempt to investigate their use as a streamline diagnostic and prognostic tool. For this purpose, specific primers were designed to amplify a genomic sequence unique to P. vivax. As expected, amplification of this genomic sequence was not detected in samples from healthy donors   .It is reasonable to speculate that parasite specific DNA is present among the CNAs circulating in plasma. To confirm this, we assessed the levels of y donors . Further 0.0497) , they we 0.2056) . Also, pP. vivax malaria morbidity. We show here that CNAs levels in plasma from P. vivax patients increase linearly with the clinical spectrum of the disease. This confirms that this powerful marker can also be used in malaria as a sensitive indicator of inflammation and injury. In fact, plasma CNAs levels have been regarded as a noninvasive universal cancer biomarker This study is the first to investigate the use of plasma levels of cell-free circulating nucleic acids (CNAs) as a marker of P. vivax malaria as their levels are often associated with malaria clinical manifestations. Nevertheless, we believe that CNAs offer a more sensitive tool since qPCR amplification of hTERT, a specific single copy human genomic sequence, revealed that levels as low as 100 fentogram of CNAs could be detected circulating in plasma, and were able to discriminate different degrees of disease morbidity  is a common hematological finding in patients with Plasmodium infection particularly in vivax malaria P. vivax in comparison to either P. falciparum monoinfection or mixed infections Thrombocytopenia  are associated with the clinical spectrum of disease, including fever and prolonged time with malaria symptoms In uncomplicated P. vivax patients. Although our results revealed that host and parasite sequences are part of the total plasma CNAs levels in acute P. vivax infected patients, the levels of a host specific (hTERT) but not parasite specific sequence correlated with vivax clinical disease. These results are in agreement with a recently study carried out in the Amazon area in which high parasitemia was not the rule among patients with severe disease according to the WHO criteria To investigate if parasite derived-sequences are part of the pool of nucleic acids circulating in blood during vivax malaria, and if these sequences correlate with disease morbidity, we assessed the levels of a parasite specific single copy genomic sequence in CNAs purified from Whether CNAs are merely inert debris of cellular injury, or if they possess pro-inflammatory properties and are, therefore, players in the immunopathogenic basis of malaria requires further investigation. Although at this point is not possible to draw conclusions, their role in the inflammatory response during malaria cannot be rule out. In fact, it is well known that dying cells spill their content and release a myriad of endogenous pro-inflammatory danger signals, including proteins, nucleic acids, extracellular matrix components, lipid mediators and adenosine triphosphate (ATP) P. vivax malaria clinical spectrum, and may have a role in malaria-induced inflammation. Given the enormous economic scourge of P. vivax in endemic areas, plasma CNAs levels provide a welcome prognostic tool to rapidly identify potentially severe cases and improve clinical management.In conclusion, we show that host circulating nucleic acids in plasma constitute a reliable and non-invasive biomarker to evaluate vivax malaria morbidity. CNAs levels were closely associated with P. vivax malaria cases in Latin America This study was conducted in May 2010, at Fundação de Medicina Tropical Dr. Heitor Vieira Dourado (FMT-HVD), a tertiary care center for infectious diseases in Manaus , the capital of the state of Amazonas, Brazil. Manaus is clearly part of a new frontier in the economic development of the Amazon and is considered as one of the leading cities in terms of number of P. vivax were invited to participate in the study. Exclusion criteria included: (i) refuse or inability to sign the informed consent; (ii) age <18 years; (ii) pregnant women; (ii) mixed infection with P. falciparum or P. malariae; (iv) any other co-morbidity that could be traced. Twenty-one patients, aging 21 to 72 years, were enrolled in the study. Selected volunteers were all negative for P. falciparum and/or Plasmodium malariae infection by both microscopic examination and a nested-PCR, carried out latter in our laboratory. Clinical and demographical data were acquired through a standardized questionnaire, and the hematological profiles were assessed by automated complete blood count carried out at FMTA hematology facility. P. vivax infected-volunteers.Individuals who sought care at FTM-HVD and whose thick blood smear was positive for The study was approved by the Ethical Review Board of the René Rachou Research Center, FIOCRUZ, Brazilian Ministry of Health (Reporter CEPSH/CPqRR 05/2008). All participants were instructed about the objectives of the study and signed an informed consent in accordance with guidelines for human research, as specified by the Brazilian National Council of Health (Resolution 196/96). Patients diagnosed with vivax malaria were treated according to the standard protocols recommended by the National Malaria Control Program (chloroquine plus primaquine).P. vivax patients  from a second hospital of the Amazon area, Julio Muller Hospital, Cuiaba, MT, which was located about 1500 miles from Manaus city. CNAS levels were also evaluated in plasma samples from a small group of P. falciparum patients . Plasma samples were isolated immediately after blood sampling and stored at −80°C until use.Peripheral blood samples (10 mL in EDTA) were obtained from patients on admission and, in those who attended follow-up, during convalescence 7 days later. Plasma samples from 14 age-matched malaria-unexposed donors from Belo Horizonte, a malaria free area, were used as baseline control. Aiming to avoid bias of selection, we further include an additional group of P. falciparum malaria P. vivax malaria patients, we adapted the criteria originally described by Karunaweera et al Since at present no clear criteria define vivax malaria severity, the present study used the World Health Organization standard criteria built for P. vivax patients or healthy donors with QIAamp Circulating Nucleic Acid Kit  according to the manufacturer's instructions. Two different methodologies were used to quantify CNAs levels in plasma: (i) amplification of the genomic sequence of the human telomerase reverse transcriptase (hTERT), an ubiquitous single copy gene mapped on 5p 15.33, used here as a marker of the total amount of DNA present in plasma samples. For that, we used the following specific primers Fw: 5′GGC ACA CGT GGC TTT TCG 3′; Rev: 5′ GGT GAA CCT GCT AAG TTT ATG CAA 3′, previously described Oncorhynchus keta) Y-chromosome specific marker was amplified in parallel with hTERT using the specific primers: Fw: 5′ AGG CAA CCC TTG CTC GAA TT 3′; Rev 5′ TGG GCA CAT GGC TTA CCG 3′; (ii) total dsDNA levels in plasmas were also quantified fluorometrically using the Quant-iTTM Pico Green Reagent  according to the manufacturer's instructions.Cell-free circulating nucleic acids (CNAs) were isolated from plasma from 5′ CAA CAG GTC CTT CAC GCT TAG TG 3′; Rev: 5′ CGA CAG CAC CAT TGG CG 3′ was designed based on the P. vivax genomic sequence http://plasmodb.org/plasmo/). The Primer Express software (PE Applied Biosystems) was used for primer design. Quantitative PCR reactions were carried out in an ABI Prism 7000 Sequence Detection System SDS . The temperature profile was 95°C for 10 min followed by 40 cycles of denaturation at 95°C for 15 s and annealing/extension at 60°C for 1 min. The cycle threshold for DNA quantification was set to 0.2 for all experiments in this study.To identify parasite derived sequences in plasma samples from infected patients the following primer pair Fw: Data were analyzed using GraphPad Prism version 5.00 for Windows . Differences in the means were analyzed using two-tailed student's t test or Mann-Whitney test when data did not fit a Gaussian distribution. Spearman nonparametric correlation coefficient was used to analyze the association between the variables.Figure S1Absolute quantification of hTERT levels in plasma from P. vivax patients. The human genomic sequence of hTERT was amplified by PCR using the primers described in M&M. The concentration of the PCR product was determined spectrophotometrically using Nanodrop. (A) A standard curve was built by re-amplifying known amounts of the hTERT PCR product in 10-fold serial dilutions. (B) Amplification of hTERT in CNAs samples purified from healthy donors or malaria patients. (C) Results of interpolated hTERT concentrations in CNAs samples purified from plasma of healthy donors or malaria patients. Levels are expressed as pg/ml. Differences were calculated by the Mann-Whitney test. A p value<0.05 was considered significant.(TIFF)Click here for additional data file.Figure S2Plasma CNAs levels correlates with vivax thrombocytopenia in a different Brazilian endemic area, Cuiaba, Mato Grosso. Correlation of plasma CNAs levels with platelet counts in 14 symptomatic vivax malaria patients attended at the hospital Julio Muller, Cuiaba, MT. (A) The mean cycle threshold for hTERT amplification was plotted against the platelet counts . (B) Assessment of CNAs levels and mean cycle threshold for hTERT amplification in samples from 10 out of 14 patients who returned after 7–10 days post treatment.(TIFF)Click here for additional data file.Figure S3Plasma CNAs levels correlates with thrombocytopenia in P. falciparum patients. CNAs levels were assessed in plasma from 9 samples from P. falciparum patients and correlated with (A) their platelet counts and (B) body temperature measured at the time of blood collection. Fluorometric dsDNA measurement by PicoGreen and qPCR amplification of hTERT genomic sequence were used for comparisons.(TIFF)Click here for additional data file.Table S1Patient final clinical score and plasma CNAs levels.(DOC)Click here for additional data file.


WHO guidelines for the treatment of young children with suspected malaria have recently changed from presumptive treatment to anti-malarial treatment guided by a blood slide or malaria rapid diagnostic test (RDT). However, there is limited evidence of the safety of this policy in routine outpatient settings in Africa.Children 3-59 months of age with a non-severe febrile illness and no obvious cause were enrolled over a period of one year in a malaria endemic area of Tanzania. Treatment was determined by the results of a clinical examination and RDT result, and blood culture and serum lactate were also collected. RDT-negative children were followed up over 14 days.Plasmodium falciparum DNA at enrolment. In addition, 12 (1.2%) children were admitted to hospital, one with possible malaria, none of whom died. A bacterial pathogen was identified in 9/965 (0.9%) children, eight of whom were RDT-negative and one was RDT-positive, but slide-negative. Excluding three children with Salmonella typhi, all of the children with bacteraemia were ≤12 months of age. Compared to double-read research slide results RDTs had a sensitivity of 97.8% (95%CI 96.9-98.7) and specificity of 96.3% (95%CI 96.3-98.4).Over the course of one year, 965 children were enrolled; 158 (16.4%) were RDT-positive and treated with artemether-lumefantrine and 807 (83.4%) were RDT-negative and treated with non-anti-malarial medicines. Compared with RDT-positives, RDT-negative children were on average younger with a lower axillary temperature and more likely to have a history of cough or difficulty in breathing. Six (0.6%) children became RDT-positive after enrolment, all of whom were PCR-negative for Use of RDTs to direct the use of anti-malarial drugs in young children did not result in any missed diagnoses of malaria although new infections soon after a consultation with a negative RDT result may undermine confidence in results. Invasive bacterial disease is uncommon in children with non-severe illness and most cases occurred in infants with a current fever. Plasmodium falciparum, the need for more reliable surveillance data as malaria has declined in many areas of Africa, and the relative neglect of alternative diagnoses to malaria [The features of malaria overlap with those of other common illnesses and, where diagnosis is based on clinical history and examination alone, this often results in a large degree of overtreatment for malaria . For man malaria -7.In parallel to this, accurate and affordable rapid diagnostic tests for malaria (RDTs) have become available over the last decade  and the This study was conducted in a malaria endemic area of Tanzania to document the clinical outcomes and blood culture results of children with febrile illness whose treatment had been guided by the results of a commonly used RDT.The study was conducted in the mother and child health (MCH) clinic of Teule Hospital, the District Designated Hospital of Muheza District, Tanzania. This serves a predominantly rural population exposed to perennial transmission of malaria with seasonal peaks coinciding with the short and long rainy seasons. In common with much of East Africa, malaria incidence has declined in recent years; in1990 parasite prevalence in children under the age of five years was approximately 80% and this had declined to 30% in 2008 ,12. In oAll children presenting to the MCH clinic were screened for the following inclusion criteria; age 3-59 months and resident within the study area, reported fever in the previous two days or current axillary temperature ≥37.5 C, no obvious non-malarial cause of fever and no known chronic illness. Children with evidence of severe illness were admitted to the paediatric ward and excluded from the study.P. falciparum .Following consenting procedures, a medical history and clinical examination were undertaken according to IMCI guidelines. Venous blood was drawn for culture (2-5ml), haemoglobin concentration , serum lactate , HRP-2 based RDT for Giemsa-stained blood slides were prepared from venous blood and independently double-read, with discordant results (either positive/negative discordance or > 2-fold density difference above 400 parasites/μL) resolved by an independent third reading. Microscopists were blinded to RDT results. Parasite densities were calculated from the geometric mean of the two closest counts of asexual parasites/200 white blood cells assuming 8,000 WBC/μl. Blood for culture was inoculated into a BactALERT™ Paediatric-fan bottle  and incubated in the BacT/ALERT 3D automated microbial detection system. Cultures that flagged positive were identified by standard methods. Cultures positive for non-cryptococcal yeasts, coagulase-negative Staphylococcus, Micrococcus, Corynebacterium, Bacillus sp. or viridans group Streptococci were considered contaminants and classified as 'negative culture' unless a pathogenic organism was also isolated. The laboratory participated in a bacteriology external quality assurance programme coordinated by a reference laboratory in Moshi, Tanzania that participates successfully in external quality assurance programmes of the College of American Pathologists. At all visits, a malaria RDT, blood slide and blood spot for parasite DNA identification by polymerase chain reaction (PCR) were obtained according to established methodology .Children were treated according to IMCI guidelines but anti-malarials or other medication with anti-malarial activity was avoided in RDT-negative children. RDT-negative children were seen and checked on Days 2, 7 and 14 following the initial visit (Day 0) with home visits the following day (and up to 2 more days until seen) if they did not attend. RDT-positive children were treated with artemether-lumefantrine (ALu) as recommended in the Tanzanian Guidelines for Malaria Diagnosis and Treatment (2006). RDT-positive children were not routinely followed up but results of positive blood cultures were promptly reviewed by a study clinician and, if the isolate was judged to be a pathogen, home visits were conducted for three consecutive days or until the child was seen and appropriate treatment and follow up was arranged. Children were also seen at additional times on parental demand and admissions to the hospital ward for children in the study were identified and clinical details obtained from hospital records.Data were double-entered in MS Access and analysed in Stata-11. Simple proportions and confidence intervals were calculated for prevalence of pathogens, and risk of malaria in RDT-negative cases at follow-up. Sensitivity and specificity of Paracheck against the gold standard of double-read research miscroscopy was undertaken on baseline samples. Groups were compared using Wilcoxon rank-sum test for continuous variables and chi-square test for proportions. The study was able to detect prevalence of pathogens in 800 RDT-negative patients at 1% with 95%CI 0.5-2% and a 5% prevalence with a 95%CI 3.6-6.6%.Written informed consent was obtained from the parent or caretaker of each child, or witnessed verbal consent if the parent could not read. The study was approved by the Ethical Review Boards of the National Institute for Medical Research in Tanzania (NIMR/HQ/R.8a/Vol.IX/785) and the London School of Hygiene & Tropical Medicine in the UK (LSHTM ethics # 5523).The study was conducted over one year from April 2009; 2592 children were screened, and 965 were eligible and enrolled Figure . Of thesRDTs had a sensitivity of 98.6% (95%CI 97.8-99.3) and specificity of 97.3% (95%CI 96.3-98.4) when compared to double-read research slide results; 14.3% of blood slides were positive with a negative predictive value of RDT results of 99.8% (95%CI 99.4-100).P. falciparum DNA at enrolment. Twenty children were lost to follow-up of whom 6 (30%) were untraceable and 14 (70%) had left the study area but were reported (by telephone or friends) to be well at the time they exited the study. In addition, 12 children were admitted to hospital for whom details are provided in Table Of the 807 patients who were RDT-negative, 765 (94.8%) remained in the study for the 14 days of follow-up at which time 461 (60.3%) of whom were reported by the caretaker to be 'completely well'. Of 42 patients who exited before the end of the study, the parents of 6 children administered an anti-malarial drug during the 14-day follow-up period and all of these were RDT-negative at their next research visit and a further six children became RDT-positive after enrolment and were treated with ALu, two of whom were slide-positive with 16,676 and 1,234 asexual parasites/μl respectively and all of whom were PCR-negative for Diagnoses made by the study clinician at the first consultation are shown in Table S. typhi, all of the children with bacteraemia were 12 months of age or youngerA bacterial pathogen was identified from blood culture in 9/965 (0.9%) children in the study, 8 of whom were RDT-negative and one was RDT-positive but slide-negative Table . In addiWhile age, haemoglobin level and presence of respiratory symptoms provide some guide to diagnostic probability, clinical predictors of malaria are still insufficient to guide treatment. The advent of RDTs provides the potential to target anti-malarial treatment on a scale that has hitherto been impossible. However, doubts remain in the minds of many clinicians about the safety of not treating parasite-negative patients for malaria, and these have probably contributed to the observation from a number of studies in Africa that up to half of those with a negative rapid test or blood slide result for malaria are treated with anti-malarials anyway. Additionally the question 'if it is not malaria what is it?' is important for framing a rational diagnostic and treatment strategy. Appropriate use of anti-microbial drugs is a key issue given the high mortality of children admitted to hospital with invasive bacterial disease but whose treatment is restricted by high levels of resistance to commonly used agents .et al used expert microscopy to guide the treatment of 2,359 illness episodes in children under the age of 10 years in a malaria-endemic area of Uganda; fewer than 1% of initial blood slide readings were false-negatives, and only 13 (0.8%) of 1,602 slide-negative illness episodes became positive over the following even days, and none of these was severely ill [et al followed up 603 RDT-negative children who had not been treated for malaria and only three children developed a positive result in the following week, all of whom were successfully treated [This study adds to other evidence that anti-malarial treatment based on parasitological testing misses few true cases of malaria. Njama-Meyer rely ill . D'Acrem treated .Although the study results are reassuring, the policy of RDT-directed treatment for suspected malaria represents a substantial change in the delivery of routine care in Africa and some questions still remain. Firstly, it is impossible to distinguish between 'new' or 'missed' diagnoses of malaria during routine follow-up of RDT-negative patients. As observed in the current study, a significant minority of children will be admitted for malaria within days of being seen with a non-severe febrile illness and a negative RDT result. Although the PCR primers used in this study were not the most sensitive, the results suggest that the malaria diagnoses following enrolment into the study were 'new' infections and that it is rational and safe to treat RDT-negative children for a non-malarial illness. However, the perception of clinicians and parents may be that a child with malaria has been denied the best treatment as a result of application of new guidelines with RDTs. Given evidence of a strong preference for a diagnosis of malaria among parents and clinicians , such anThe commonest indication for anti-microbial treatment in our study was the IMCI diagnosis of non-severe pneumonia. While the overlap between malaria and pneumonia has been previously described both in severe and non-severe illness, the proportion of children meeting WHO criteria for non-severe pneumonia was higher than expected from previous studies ,19-21. PAs new guidelines and RDTs are introduced across Africa it is possible that there will be a compensatory over-use of the IMCI diagnosis of non-severe pneumonia but this will be difficult to assess since there is no gold standard definition of the diagnosis of pneumonia. Even of the true pneumonias some will be viral, including the vaccine-preventable RSV pneumonias, where antibiotics will make little positive impact on outcome ,24. In aConsistent with at least one other study, we found that bacteraemia was uncommon in children with non-severe illness, especially in children who were RDT-positive . HoweverThis study has a number of inevitable limitations. The study was conducted in hospital outpatients where the patient population may differ in a number of ways from those attending primary health facilities generally. The accuracy of RDT interpretation may have been higher than under routine practice, and certainly higher than routine microscopy in some clinics in the region. Clinicians were aware that they were being observed (in the sense that their findings and prescriptions were being recorded) and may have been more likely to follow IMCI guidelines and to prescribe antibiotics, thus over-estimating antibiotic usage that would occur under similar conditions in routine practice. The clinical care provided in the study followed IMCI guidelines and did not have access to an otoscope or urine examination resulting in possible neglect of otitis media or urinary tract infections. The lack of validating investigations for the IMCI diagnosis of pneumonia has already been discussed.In conclusion, this study demonstrates that use of RDT's to direct the use of anti-malarial drugs in young children is a safe strategy and did not result in any missed diagnoses of malaria. However, new episodes of malaria do occur within a short period of an initial consultation in malaria endemic areas and these may undermine confidence in parasitological testing for malaria. Invasive bacterial disease is relatively uncommon in children with non-severe illness; while most cases occurred in infants with a current fever there is a need for more specific diagnostic tests to detect these infections at the point of care.The authors declare that they have no competing interests.GM was responsible for clinical data collection and drafting the MS, IH was responsible for drafting study documents and supervising data collection, BA was responsible for the laboratory work and provided critical review of the MS, HM was responsible for data collection, VM was responsible for clinical care, AM was responsible for the PCR analysis, FM assisted in the analysis and provided critical review of the MS, AS contributed to the design of the study and provided critical review of the MS, HH assisted in the analysis and writing and provided critical review of the MS, CW initiated the idea for the study and contributed to the writing of the MS, HR was involved in the design of the study, analysed the data and co-wrote the MS. All authors read and approved the final manuscript.


Plasmodium falciparum remains the principle public health problem for the population. In 2010, Médecins Sans Frontières (MSF) implemented an innovative multi-component malaria intervention in Guéckédou prefecture, south-western Guinea. The objective of the intervention is to decrease morbidity, mortality and transmission of malaria by reinforcing health facilities and ensuring access to malaria tests and treatment. The impact of the intervention is measured by a series of cross sectional surveys.The Republic of Guinea (Guinea) has year round malaria transmission with a seasonal peak corresponding with the rainy season. According to both programmatic and health center data, malaria caused by In April 2011 the first of seven cross sectional surveys was carried out in four strata; two rural areas and one urban area with MSF intervention and one rural area with limited MSF support. Within each strata probability proportionate to size cluster sampling was used to select clusters. Households were randomly selected in each cluster and all permanent residents invited to participate. Informed consent was obtained from all participants or their guardian. In addition to administering a standardized questionnaire and carrying out a brief clinical exam, a blood slide with both thick and thin films was made and each participant was tested for malaria using SD Bioline HRPII rapid test regardless of their symptoms. All participants who tested RDT positive on the day of the survey were treated with the first line malaria treatment according to the National Malaria Control Protocol or referred to the nearest health facility.Plasmodium falciparum infection. Further laboratory quantification revealed that 11.1% of infected individuals had levels of parasitemia within the lower limits of RDT detection, 50-100 parasites/µl and 18.5% of microscopy positive individuals had parasitemia below the RDT detection threshold, ≤50 parasites/µl and were RDT negative.Over 6,800 people participated in this survey. Children <5 years of age constituted 31% of the survey sample, children 5-14 years of age 27%, and individuals ≥15 years 42%. Women comprised 56.1% of the surveyed population and men 43.9%. A total of 4788 available samples were positive for malaria with microscopy for a point prevalence of 68.6%  while 61.3% of individuals were identified as positive with RDT . 99.5% of microscopically diagnosed malaria infections were due to Prior to our survey, aside from health facility estimates, the malaria burden in and around Guéckédou was unknown. Laboratory analysis demonstrates that the gold standard, microscopy, remains the most reliable method to detect malaria infections. Additionally, exclusive diagnosis with malaria rapid tests similar to those currently on the market will not identify all parasitaemic individuals. In Guéckédou, a hyper endemic context, 18.5% of infected individuals were not identified when only using RDTs for diagnosis. In areas targeted for elimination where a significant proportion of the population has low level parasitemia the use of rapid diagnostic tests alone is not sufficient to detect and thus eliminate the parasite reservoir.


Although severe malaria is an important cause of mortality among children in Burkina Faso, data on community-acquired invasive bacterial infections  are lacking, as well as data on the involved pathogens and their antibiotic resistance rates.The present study was conducted in a rural hospital and health center in Burkina Faso, in a seasonal malaria transmission area. Hospitalized children (<15 years) presenting with T≥38.0°C and/or signs of severe illness were enrolled upon admission. Malaria diagnosis and blood culture were performed for all participants, lumbar puncture when clinically indicated. We assessed the frequency of severe malaria  and IBI, and the species distribution and antibiotic resistance of the bacterial pathogens causing IBI.Salmonella spp. (NTS) was the predominant isolate from blood culture (32.8%), followed by Salmonella Typhi (18.8%), Streptococcus pneumoniae (18.8%) and Escherichia coli (12.5%). High antibiotic resistance rates to first line antibiotics were observed, particularly among Gram-negative pathogens. In addition, decreased ciprofloxacin susceptibility and extended-spectrum beta lactamase (ESBL) production was reported for one NTS isolate each. ESBL production was observed in 3/8 E. coli isolates. In-hospital mortality was 8.2% and case-fatality rates for IBI (23.4%) were significantly higher compared to severe malaria .From July 2012 to July 2013, a total of 711 patients were included. Severe malaria was diagnosed in 292 (41.1%) children, including 8 (2.7%) with IBI co-infection. IBI was demonstrated in 67 (9.7%) children , 8 (11.8%) were co-infected with malaria. Non-Typhoid Although severe malaria was the main cause of illness, IBI were not uncommon and had higher case-fatality rates. The high frequency, antibiotic resistance rates and mortality rates of community acquired IBI require improvement in hygiene, better diagnostic methods and revision of current treatment guidelines. Malaria remains a major cause of mortality in children in most sub-Saharan African countries, despite decreasing incidence rates Neisseria meningitidis A was performed Haemophilus influenzae type b was introduced into EPI in January 2006 and the estimated coverage of immunization was 95.2% in 2011 The survey was conducted at the district hospital Centre Médical avec Antenne chirurgicale (CMA) Saint Camille of Nanoro and the health center Urbain (with inpatient beds), both situated in Nanoro, Burkina Faso. Nanoro is located in a rural area in the Center-West region of the country, approximately 90 km west of the capital Ouagadougou. The health district of Nanoro comprises approximately 153,259 inhabitants of whom 35,159 (22.9%) are children <5 years of age All admitted children <15 years of age presenting with axillary temperature ≥38.0°C or clinical signs of severe illness were enrolled, including respiratory distress, prostration, altered consciousness, convulsions, clinical jaundice, hypothermia, signs of shock or severe malnutrition  or with severe anemia (hemoglobin <5 g/dl). Medical history, including prior antibiotic (48 h) and antimalarial (2 weeks) treatment, physical examination and outcome of febrile episode  were registered on a standardized form by trained study nurses. Venous blood samples for blood culture, malaria diagnosis, full blood count and blood glucose levels were collected from all participants by trained study staff using aseptic techniques. Lumbar puncture was performed in case of suspicion of cerebral malaria and/or bacterial meningitis, based on the decision of the attending health care worker. Decision for HIV testing or other additional testing was left to the discretion of attending health staff. Treatment decisions were made by the attending health staff according to national guidelines: quinine IV for severe malaria, ampicillin + gentamicin for neonatal infections and ceftriaxone for suspected sepsis or meningitis in older children. Laboratory results were provided instantly to guide treatment decisions.All laboratory analysis were performed at the Clinical Research Unit of Nanoro (CRUN) which is located on the compound of CMA. Samples were submitted within 15 minutes after collection.Plasmodium species according to standard procedures Plasmodium species, difference in parasite density >Log10 or ratio >2 in case of parasite density ≤400/µl and >400/µl respectively) by a third experienced microscopist. A selection of slides (5%) was re-read by an expert microscopist whose results were considered conclusive.Thick and thin blood films were stained with 3% Giemsa solution (pH 7.2), examined for presence of P. falciparum-specific histidine-rich protein-2 (PfHRP2), was performed on EDTA blood samples according to the manufacturers' instructions.The malaria rapid diagnostic test (RDT) recommended by the national malaria control program, SD Bioline Pf  detecting Salmonella spp., multidrug resistance (MDR) was defined as resistance to the first-line antibiotics ampicillin, chloramphenicol and trimethoprim-sulfamethoxazole (TMP-SMX); reduced fluoroquinolone susceptibility  was defined as resistance to nalidixic acid Enterobacteriaceae resistant to cephalosporins were tested for Extended Spectrum Beta-Lactamase (ESBL) production by combined double-disk method  nterobacteriaceae, no breakpoints have been published for azithromycin susceptibility, however EUCAST V 3.1. Salmonella Typhi at MIC-values ≤16 mg/l and recently this value has been proposed as an epidemiological cutoff value for wild-type Salmonella spp. Shigella spp. and a subset of Salmonella spp. isolates was confirmed at the National Reference Laboratory for Salmonella and Shigella  by slide agglutination with commercial monospecific antisera , following the Kauffmann-White scheme In all children, 1–3 ml of venous blood was collected into a pediatric blood culture bottle . A subset of blood culture bottles was weighed before and after blood inoculation to determine the amount of blood collected. All blood culture bottles were incubated in a BACTEC 9050 instrument (Becton Dickinson) for a total of 5 days. If flagged for growth they were Gram stained, subcultured on Eosin-Methylene blue (EMB) agar and 5% Sheep Blood agar  and incubated at 35–37°C for 24 hours in atmospheric conditions and at 5% CO2 respectively. Isolates were identified to the species level by standard biochemical methods and antibiotic susceptibility testing was performed by disk diffusion according to CLSI criteria 2 for 48 hours. Any bacterial growth observed was further processed by standard microbiological techniques. All CSF samples were assessed by latex agglutination testing for Haemophilus influenzae b, Streptococcus pneumoniae, Streptococcus group B, Neisseria meningitidis and Escherichia coli K1 . In addition if N. meningitidis was identified serotyping was performed (BioRad or Becton Dickinson).Cerebrospinal fluid (CSF) was assessed for its aspect, cell count, Gram stain and culture. The CSF leucocyte count was assessed in a Nageotte counting chamber at ×400 magnification, and if ≥50 WBC/µl were counted a thin film was prepared and stained with methylene blue to count the percentage of neutrophils and lymphocytes. For culture, 1 to 2 drops of CSF were inoculated onto blood agar and chocolate blood agar supplemented with Isovitalex™ (Becton Dickinson) and incubated at 35–37°C at 5% COFull blood counts were assessed using Sysmex XS1000i .For determination of glucose levels full blood was collected on NaF tubes and processed by Flexor Junior . Screening for HIV was performed at the HIV center on the CMA compound according to national guidelines by the rapid diagnostic test Determine™ HIV-1/2  and in case positive the SD Bioline HIV-1/2 3.0  was performed.Fever was defined as axillary temperature ≥37.5°C or reported history of fever in the past 48 hours.P. falciparum parasites confirmed by microscopy.Malaria was defined as the presence of asexual Severe malaria was defined as microscopically confirmed malaria and fulfillment of at least one of the WHO clinical or laboratory criteria of severe malaria falciparum malaria with coma  or convulsions in the past 24 hours  and exclusion of bacterial meningitis or hypoglycemia alone (prompt recovery of consciousness after glucose infusion) as cause of symptoms Cerebral malaria was defined as severe Staphylococci, Bacillus spp. and Micrococcus spp.IBI was defined as bacteremia and/or bacterial meningitis. Bacteremia was defined as the growth of clinical significant organisms from blood culture. Non-pathogenic bacteria or skin flora were considered contaminants, including coagulase-negative Bacterial meningitis was defined as i) cerebrospinal fluid (CSF) culture grown with an organism of known clinical significance, or ii) CSF white blood cell count >10/µl with on Gram stain slides the presence of organisms of known clinical significance, or iii) CSF white blood cell count >10/µl and blood culture grown with an organism of known causative role in meningitis or iv) positive latex agglutination. Probable meningitis was defined as a child with clinical signs of meningitis, in whom no lumbar puncture had been performed and a bacteria known to be a causative agent in meningitis was isolated from blood culture.Clinical and laboratory data was double entered in Epi Info version 3.5.3. Statistical analysis was done with Stata 11 .Categorical data were assessed for significance using the chi-square test or Fisher exact test as appropriate. The Wilcoxon rank sum test was used for non-parametric data. Case-fatality rates (CFR) were calculated as proportions of children who died within a specified disease group. Only participants for whom the final hospital outcome was known (discharge or death) were included in the denominator of CFR calculations, referred patients were excluded.The study was conducted according to the principles expressed in the Declaration of Helsinki and was approved by the national ethics committee of Burkina Faso, the institutional review board of the Institute of Tropical Medicine, Antwerp and the ethics committee of the University Hospital of Antwerp. Written informed consent was given by all parents or guardians of children enrolled.p>0.05) between included and eligible non-included patients, however, recorded axillary temperature was significantly higher among included patients (p<0.001).From July 2012 to July 2013, a total of 1134 children were admitted to the hospital, 337 were considered non-eligible and another 177 eligible children were not included for various reasons . In total 711 admitted children were included: 607 at the hospital and another 104 children at the health center. For age, gender and outcome, there were no significant differences .In 378 children (53.2%) malaria was confirmed microscopically. In total 292 children (41.1%) fulfilled the diagnosis of severe malaria, of whom 48 children (16.4%) had cerebral malaria and 136 (46.6%) severe malaria anemia , includip = 0.799), neither when stratified by prior antimalarial treatment.Parasite density was not significantly different for those fulfilling criteria of severe malaria  and those not .Of note, a positive malaria RDT was observed in 137 children with negative microscopy, among which 106 (77.4%) had at least one clinical or laboratory sign of severe malaria. Of them, 69.8% (74/106) reported previous antimalarial treatment, significantly higher compared to children with microscopically confirmed severe malaria , Salmonella Typhi (18.8%) Streptococcus pneumoniae (18.8%) and Escherichia coli (12.5%) , Salmonella Enteritidis (n = 8) and Salmonella Telelkibir (n = 1). E. coli, NTS and Salmonella Typhi were the most frequently isolated pathogens among children <1 year of age, 1–5 years and ≥5 years of age respectively (Bacteremia was found in 63 (8.9%) children , while c (12.5%) . NTS comectively .The median (interquartile range) blood volume per blood culture vial sampled was 0.8 ml (0.3–1.2) and 2.1 ml (1.1–3.0) for children <2 months and ≥2 months of age respectively. In 15/170 (8.8%) and 10/170 (5.9%) children blood culture bottles were under-filled (<0.5 ml) and overfilled (>5.0 ml) respectively.Streptococcus pneumoniae (n = 3), Neisseria meningitidis W135 (n = 1), Haemophilus influenzae (n = 1) and Klebsiella pneumoniae (n = 1). There were five additional children with probable meningitis who had S. pneumoniae (n = 4) and N. meningitidis W135 (n = 1) isolated from blood cultures.Bacterial meningitis was confirmed in 6/19 (31.6%) children for whom lumbar puncture was performed; pathogens were P. falciparum parasites were present in 7/63 (11.1%) and 1/6 (16.7%) children with bacteremia and meningitis respectively. Conversely, among the children with severe malaria 8/292 (2.7%) had IBI. No significant difference in parasite densities between children with severe malaria  and severe malaria/IBI co-infection  was observed (p = 0.5).Distribution of positive microscopy, RDT and IBI among children included are displayed in p<0.001).The RDT was positive in 30/63 (47.6%) children with bacteremia and was significantly more frequent among children with NTS  compared to children with other pathogens isolated . All other NTS isolates were susceptible to ciprofloxacin . Salmonella Telelkibir was susceptible to all antibiotics tested. Among the Salmonella Typhi isolates, 10/12 (83.3%) were resistant to both chloramphenicol and TMP-SMX but all were susceptible to ampicillin and ciprofloxacin . Shigella flexneri was resistant to ampicillin, chloramphenicol and TMP-SMX, Shigella dysenteriae was resistant to ampicillin and TMP-SMX. All NTS, Salmonella Typhi and Shigella isolates were susceptible to azithromycin . Three E. coli isolates were confirmed ESBL producers and co-resistant to ciprofloxacin, two of them were also co-resistant to gentamicin. No resistance to the carbapenem antibiotics tested was detected.S. pneumoniae isolates  were susceptible to ceftriaxone  and 10/11 were susceptible to penicillin . The remaining isolate had MIC-values (0.094 µg/ml) above the CLSI meningitis susceptibility breakpoint (0.06 µg/ml). All S. pneumoniae isolates were susceptible to erythromycin but most 9/11 (81.8%) were resistant to TMP-SMX. The N. meningitidis isolates were susceptible to penicillin  and ceftriaxone  as was H. influenzae All S. aureus isolates were methicillin susceptible as well as susceptible to clindamycin, gentamicin and TMP-SMX; three and one of them were resistant to tetracycline and erythromycin respectively.All three p = 0.015).A total of 28.2% (205/728) children reported antibiotic use <48 h prior to sampling; most frequently administered antibiotics were TMP-SMX (35.1%) and amoxicillin or ampicillin (45.9%). Bacteria were significantly more frequently isolated among children on antibiotics  compared to those who reported no prior antibiotic use , none of the children with severe malaria/IBI co-infection (n = 8) died. Among children with a positive RDT and negative microscopy with signs of severe malaria 11/110 (10.0%) died, of them six had IBI.For 14 children the final outcome could not be ascertained because they were referred to a tertiary care center (n = 8) or left the hospital before completion of treatment (n = 6). For 697 (98.0%) children hospital outcome was known: 58/697 (8.3%) children died, of them 20 (34.5%) and 15 (25.9%) were diagnosed with severe malaria and IBI respectively. CFR were significantly higher among children with IBI (23.4%) versus severe malaria , E. coli (4/8), NTS (3/20), S. aureus (2/3) and N. meningitidis (1/2). None of the patients with Salmonella Typhi bacteremia died. Among children with confirmed and probable meningitis 4/10 (40.0%) died , while 8/46 (17.4%) children with cerebral malaria died.CFR for IBI was highest among children <1 year of age . Among children with bacteremia, deaths occurred among children infected by The present study showed that, among children ill enough to be admitted to rural health facilities in Burkina Faso, severe malaria was the leading cause of morbidity (40% of admissions) and mortality (one third of deaths). IBI were however not uncommon and were associated with a significantly higher CFR compared to malaria. The relative proportions of severe malaria and IBI in febrile and/or severely ill children differed dramatically according to the seasons as well as the bacterial pathogens isolated, with a peak of severe malaria during the rainy season and a relative increase of IBI (mainly due to NTS) shortly thereafter. High resistance rates to first line antibiotics were observed, particularly among Gram-negative pathogens.e.g. 40–80% in the case of typhoid fever S. pneumoniae, may have been missed. Quality indicators of the current blood culture system were satisfying: the percentage of contaminants (3.7%) was acceptable and lower compared to other studies performed in malaria endemic settings (8–14.3%) P. falciparum parasites, which is not uncommon in Burkina Faso This study has several limitations. First, a substantial proportion of children fulfilling inclusion criteria was not included. As age and mortality rate did not differ significantly, non-inclusion probably occurred at random although the lower body temperatures may suggest less severe disease in the non-included patients. Second, blood cultures intrinsically have moderate sensitivity  Bacterial co-infections among children with severe malaria were less frequent (2.7%) compared to those reported from other studies (4.6%–8.3.%) Salmonella Typhi was relatively frequent in our study compared to others performed in similar settings Salmonella Typhi is more common in older children Salmonella Typhi bacteremia have reported to be even higher among children 2–5 years compared to children ≥5 Except for surveillance of meningitis epidemics and a single study on blood culture isolates in a tertiary care urban center Enterobacteriaceae. The high resistance rates to the first line antibiotics TMP-SMX and amoxicillin/ampicillin requires extension of surveillance studies to other populations  and places in the country and revision of the current national treatment guidelines. Of concern are the findings of DCS and ESBL in one NTS isolate each and especially the presence of ESBL with co-resistances to gentamicin and fluoroquinolone antibiotics among E. coli, precluding treatment options with commonly available antibiotics. All S. pneumoniae, N. meningitidis and H. influenzae isolates were susceptible to ceftriaxone, which is the recommended treatment for bacterial meningitis in Burkina Faso.Resistance rates among community-acquired pathogens were high, particularly among the CFR for IBI were significantly higher compared to severe malaria (23.4% and 6.8% respectively) and existence of IBI as well as appropriate case management should be more emphasized at all levels of health care.As malaria may predispose to NTS bacteremia In conclusion, although severe malaria was the main cause of disease among children admitted to a rural hospital in Burkina Faso, invasive bacterial infections were not uncommon and had higher CFR. The high proportion and CFR of community acquired bacteremia as well as the high antibiotic resistance rates of the pathogens require improvement in hygiene, better diagnostic methods and emphasize the need of blood culture surveillance and revision of current treatment guidelines.


Malaria is a disease that still affects a significant proportion of the global human population. Whilst advances have been made in lowering the numbers of cases and deaths, it is clear that a strategy based solely on disease control year on year, without reducing transmission and ultimately eradicating the parasite, is unsustainable. This article highlights the current mainstay treatments alongside a selection of emerging new clinical molecules from the portfolio of Medicines for Malaria Venture (MMV) and our partners. In each case, the key highlights from each research phase are described to demonstrate how these new potential medicines were discovered. Given the increased focus of the community on eradicating the disease, the strategy for next generation combination medicines that will provide such potential is explained. Plasmodium and has historically been one of the deadliest diseases for humankind. In recent years, there has been a reduction in the numbers of deaths from malaria, and this has been attributed to the use of insecticide-treated bed-nets, indoor residual spraying to kill mosquitoes (the vector that carries the parasite and infects humans) and the availability of effective, well tolerated and high quality medicines , the first line medicines in this case, are extremely safe and effective after three days of dosing. These are medicines that combine an artemisinin derivative  with a partner drug. Artemisinin is a natural product and is extracted from the leaves of the dly WHO, . Combinifalciparum malaria due to reduced parasite sensitivity. It is now replaced by more recent analogues without cross resistance: amodiaquine, piperaquine and the ‘quinacrine-amodiaquine’ hybrid, pyronaridine. Structures of these molecules and other clinical derivatives of each class are shown in et al. The partner drugs that are used in these combinations also share their origins in nature, being synthetic variants on the natural product quinine. Broadly speaking they separate into two chemical classes: the amino-alcohols, which share the closest similarity to quinine, and the 4-aminoquinolines, which were early synthetic mimics. The amino-alcohol class is best illustrated by mefloquine and the more structurally divergent lumefantrine. Chloroquine, the original exemplar of the 4-aminoquinoline class is no longer in use for Plasmodium falciparum, the dividing or liver schizont stage lasts for around 6 days. However, for P. vivax (the key species in Asia and the Americas) and P. ovale there is also a sub-population of the liver-stage parasites, called hypnozoites, that appears to halt its development and become dormant, only to relapse after a period which can vary between four weeks and several years. Once the primary blood-stage infection has been treated, the patient is asymptomatic and often unaware of the risk associated with retention of these parasites. Although the biological mechanisms underlying the activation process are not understood, it is clear that hypnozoites can ‘wake up’ and reactivate leading to a new blood-stage infection in the absence of a mosquito bite – a relapse  is being developed for presumptive treatment and future chemoprotection in pregnancy  as highlighted from the output of the malERA consortium and is the first Target Product Profile – TPP1. Such a medicine would transform drug treatment for malaria.A second type of medicine needed in the early stages of elimination is that of chemoprotection, and is described by TPP2. Again, ideally this Single Exposure Chemoprotectant (SEC) would be able to be given on a once per month basis with the knowledge that between dosing periods individuals were protected from primary blood-stage infections.et al.P. vivax relapse (termed 3a) and gametocytes to stop transmission (termed 3b); and (4) TCP4 – Slow-onset chemoprophylactics or causal prophylactics with long duration and excellent safety profiles to protect vulnerable populations in once endemic regions.As discussed earlier, any new medicines developed should be a combination of at least two complementary molecules. Following consultations with its partners, MMV has defined four Target Candidate Profiles (TCPs), which aim to guide the identification of compounds that might be combined to produce a medicine that meets the TPPs described above , their partners and others, new molecules with novel modes of action are entering into pre-clinical development and beyond . It is now being tested in combination safety studies, and will start combination efficacy studies in 2014. Studies in both healthy volunteers and infected patients show a significant plasma exposure for as long as 20 days, suggesting it might be possible to use as part of a single-dose therapy for uncomplicated malaria  which was the first clinical candidate and which was licensed to Ranbaxy , Monash University and MMV , and this results in an increase in the concentration of sodium ions in the parasite, which is toxic to the cell.A second compound in Phase II is a novel, synthetic spiroindolone, developed by the Novartis Institute for Tropical Diseases (NITD) in Singapore in a collaboration that included the Swiss TPH, the Dutch Biomedical Primate Research Center, the Genomics Institute of the Novartis Research Foundation (GNF) and MMV. It was the first compound to be developed using whole-cell screening against the parasite  see The spiroindolone class was found from screening a library of natural product and ‘natural product like’ compounds at Novartis. The starting ‘hit’, compound in vitro and in vivo, and has been shown to kill gametocytes demonstrating potential in transmission blocking. The series acts via a novel mechanism, involving a previously unannotated gene now called P. falciparum Cyclic Amine Resistance Locus (Pfcarl)  in San Diego, delivered GNF156 as a new clinical compound, again in collaboration with MMV. The compound, an imidazolopiperazine, has high potency against blood and liver stages 3, was also identified from whole cell screening. The first task was to replace metabolically labile aromatic substituents and to stabilize likely positions of metabolism with blocking halogens. This led to compound 4 whereby potency and stability had both been improved – though not sufficiently for a candidate. Metabolite identification and excellent medicinal chemistry then led to the isomer, GNF156, whereby the metabolically susceptible position on the piperazine was blocked with two methyl groups. GNF156 demonstrated a good overall profile with an ED99 in the P. berghei mouse of 1·1 mg/kg  is known to be essential for the survival of the parasite. A team led by Meg Phillips at the University of Texas Southwestern, in collaboration with the University of Washington, Monash University and MMV identified a potent and selective triazolopyrimidine-based inhibitor DSM1 via a high-throughput enzyme screen. The three-dimensional structure of the enzyme-inhibitor complex was resolved and the subsequent lead optimization programme led to the identification of the preclinical candidate. DSM1 was an interesting molecule but with non-optimal pharmacokinetics on repeat dosing and insufficient potency. First of all, progress was made to improve pharmacokinetics by substituting electron-withdrawing groups in the aniline ring; due to the hydrophobic nature of the binding site only lipophilic groups have had major success here, e.g. DSM191  inhibitors such as pyrimethamine have been widely used for the treatment of malaria, although their clinical efficacy has been compromised by resistance. P218 is a next generation inhibitor of DHFR that has been delivered by the Thai BIOTEC group in collaboration with Monash University, the London School of Hygiene and Tropical Medicine and MMV; it has largely finished preclinical development. Impressively, the team designed P218 using structure-based design methods to have a high affinity to both the parent and mutated enzymes and to also kill both wild type and clinically-relevant resistant strains. The challenge of sustaining activity across even quadruple-mutated enzymes was overcome by starting with the pyrimethamine scaffold and then using a flexible side-chain that could adopt a variety of conformations to achieve potency such as P65. A carboxylic acid was also found to be optimal in selective binding to a key protein arginine in the parasite, but not in man see . P218 haIn the development of new treatments for malaria a number of key generic criteria need to be met by any drug candidates, in addition to those eradication criteria already discussed: high potency, low cost, high stability in tropical conditions, good development potential (e.g. acceptable solubility), a low chance for resistance developing, and high levels of safety in the target populations.in vitro method to assess these characteristics quantitatively in P. falciparum during the early phases of the drug development process has recently been developed , is also a priority. Pharmacokinetics clearly becomes the defining parameter in this case and once P. vivax is a critical gap in the malaria eradication agenda. To achieve eradication, the entire human reservoir of malaria parasites must be eliminated, including latent dormant stages. This will be particularly difficult to achieve for P. vivax because of the relapsing liver-stage hypnozoites that are refractory to treatment with all current antimalarials except primaquine.The lack of safe and easily administered drugs for radical cure of P. vivax is the inability to conduct cellular screens for anti-relapse agents. To develop a P. vivax liver-stage assay, the malaria community needs to secure a supply of viable sporozoites. Maintaining P. vivax in continuous in vitro culture to provide a clonal supply of parasites on which the mosquitoes can feed is the major barrier to this. Stable and infective hepatocyte lines are also needed, as primary human liver cells are often of variable quality and lose their differentiation in culture  and thus new compounds can be dosed once the patent blood stages have been cleared and the animal parasitaemia monitored for a relapse. It was this model that was originally used in the screening that led to the discovery of the drug primaquine and the follow-on compound, tafenoquine. However, for the development of a large drug discovery programme an in vitro model is required to facilitate high-throughput screening of compounds. Cultured hepatic stages of P. cynomolgi have been established in rhesus hepatocytes but determination of which non-dividing forms correspond to hypnozoites has proven difficult. It has been suggested that, by noting the response of these non-dividing forms to an anti-relapse drug and an antimalarial that is not active against hypnozoites, a better characterization of true hypnozoites can be obtained  under certain circumstances and this is therefore the gold standard to beat. The challenge will persist whilst this recommendation remains (and that will depend on the results of future studies), until extensive resistance to primaquine is demonstrated or, finally, hitherto unknown adverse events from the low dose become apparent. However, the unsuitability for pregnant women and very young children highlights improvements necessary in next generation molecules.et al.The strategy is to target host gametocytes such that mature gametocytes are either killed or rendered inactive. In such a scenario, following a mosquito bite parasite gamete formation is blocked either because of an absence of mature gametocytes in the blood, or their inactivation. With gamete formation inhibited in the mosquito then there cannot be further development of vector-stage parasites and hence the life-cycle is blocked. This approach reduces the transmission window for a patient and the length of this time-window is defined by the rate with which gametocytes are killed or inactivated. For primaquine this transmission window appears to be around 24–48 h . Potential additional in vivo work including a mouse-to-mouse transmission may also be instructive particularly in order to compare against primaquine, which requires metabolism for efficacy  or those that only kill liver schizonts for chemoprotection strategies.et al.Falciparum and vivax liver-stage assays are still embryonic but, ideally, it would always be important to confirm activity on the human parasite prior to significant investment in a drug discovery programme.Longer duration blood-stage assays (96 h) can be run and compared with results from earlier time points (48 h or 72 h) to prioritize compounds with slow blood-stage action (Dahl and Rosenthal, In vivo rodent models are available such as the P. berghei sporozoite infection model and these can even be performed using imaging to detect parasitaemia prior to a blood stage infection (e.g. Sanchez et al.Currently, the mainstay treatments for malaria are effective and safe. Furthermore, new clinical entities that offer significant advances over existing regimens continue to progress well and a range of compound and project options is available in Preclinical Development and earlier. However, the tenacity of the parasite in thwarting our attempts to kill it is unparalleled. If we are serious about controlling and ultimately eradicating the parasite then together we need sustained efforts to discover, develop and deliver new medicines that not only cure patients, but block transmission, block relapse and offer protection against new infections. The advent of the new MMV TPPs and TCPs will help to guide researchers towards these ends.


Plasmodium falciparum erythrocyte membrane protein 1 (PfEMP1) on the surface of parasitized red blood cells (RBCs), thereby weakening their cytoadherence to microvascular endothelial cells (MVECs) and impairing their activation of MVECs to produce pathological responses. Therefore, we hypothesized that parasites causing malaria in HbAS or HbAC heterozygotes have overcome this protective mechanism by expressing PfEMP1 variants which mediate relatively strong binding to MVECs. To test this hypothesis, we performed 31 cytoadherence comparisons between parasites from HbAA and HbAS (or HbAC) Malian children with malaria. Ring-stage parasites from HbAA and HbAS (or HbAC) children were cultivated to trophozoites, purified, and then inoculated in parallel into the same wildtype uninfected RBCs. After one cycle of invasion and maturation to the trophozoite stage expressing PfEMP1, parasite strains were compared for binding to MVECs. In this assay, there were no significant differences in the binding of parasites from HbAS and HbAC children to MVECs compared to those from HbAA children . These data suggest that in-vitro reductions in cytoadherence by HbS and HbC may not be selecting for expression of high-avidity PfEMP1 variants in vivo. Future studies that identify PfEMP1 domains or amino-acid motifs which are selectively expressed in parasites from HbAS children may provide further insights into the mechanism of malaria protection by the sickle-cell trait.Sickle hemoglobin (Hb) S and HbC may protect against malaria by reducing the expression of  Plasmodium falciparum malaria has selected for red blood cell (RBC) polymorphisms, including sickle hemoglobin (Hb) S, HbC, α-thalassemia, and G6PD deficiency 6 glu->val) is a balanced polymorphism in which HbAS heterozygotes are protected against both uncomplicated and severe malaria 6 glu->lys) generally affords protection against severe, but not uncomplicated, malaria + T cells and upregulation of heme-oxygenase 1; accelerated acquisition of immunity; and altered host pathogenic mechanisms , the parasite's variant surface antigen and cytoadherence ligand, on ‘knobs’ on the surface of parasitized HbAS and HbAC RBCs For millennia, the significant morbidity and mortality of Abnormal PfEMP1 display is characterized by (i) reduced PfEMP1 levels, (ii) reduced knob densities, (iii) heterogeneous distributions of PfEMP1 and knobs, and (iv) aberrant – wider and more protuberant – knob morphologies var genes, which encode PfEMP1 var genes Var genes share a two-exon structure, which encodes for a semi-conserved intracellular domain (exon 2) and an extracellular domain (exon 1) comprised of Duffy binding-like domains (DBLs) and cysteine-rich interdomain regions (CIDRs) The expression of PfEMP1 on parasitized RBCs also leaves parasites vulnerable to detection by the immune system. To avoid this, individual parasites undergo antigenic variation by switching from one clonally-expressed PfEMP1 variant to another Differential affinity of individual DBLs and CIDRs for specific host endothelial receptors  enables the organ-specific sequestration of parasitized RBCs and influences the clinical presentation and severity of malaria in-vivo studies to validate in-vitro findings. In considering ways to investigate these potential mechanisms, we hypothesized that parasites causing clinical malaria in HbAS and HbAC children must have somehow overcome the malaria protective effects of abnormal PfEMP1 display. One potential mechanism is that parasite populations in HbAS and HbAC children are selected to express particular PfEMP1 variants, which mediate stronger binding to MVECs in vivo than those expressed in HbAA children. This process could enable parasites to circumvent the protective effects of abnormal PfEMP1 display and cause malaria symptoms in HbAS and HbAC children. To explore this possibility, we tested whether parasites from HbAS and HbAC children with malaria show increased binding to MVECs in vitro than those from HbAA children in Mali.One challenge in defining the mechanisms underlying malaria protection in HbS and HbC children is the difficulty of conducting human P. falciparum-infected RBCs and uninfected RBCs were obtained from an ongoing cohort study conducted in three villages  located 75 km southwest of Bamako, Mali. From these villages, 1514 children aged 0.5–17 years were enrolled over three transmission seasons . All children were typed for Hb polymorphisms , ABO blood group (agglutination assay), α-thalassemia (nested PCR), and presence of the G6PD deficiency allele, G6PD*A- (G202A) (nested PCR), as described G6PD*(A-) allele] from healthy volunteers were similarly collected, washed, resuspended at 50% hematocrit, and stored at 4°C for up to 3 days before use. Ring-stage parasites from HbAA, HbAS, and HbAC children with malaria  and inoculated into wildtype RBCs at 2.5% hematocrit. After invasion of uninfected RBCs was confirmed by the appearance of new ring-stage parasites, the hematocrit was decreased to 0.8%. Once parasites matured to trophozoites, they were assayed for cytoadherence to MVECs in parallel with one or more other parasite isolates that were simultaneously cultivated, purified, and inoculated into the same wildtype RBCs.Venous blood samples from children with malaria were collected in sodium heparin Vacutainers , and the RBCs washed three times in RPMI 1640 . Uninfected ‘wildtype’ donor RBCs [i.e., HbAA, normal αα/αα genotype, and no  malaria Table 1 Human dermal MVECs  were grown in the manufacturer's EGM-2MV media for up to 5 passages and seeded into 8-well LabTak CC2-coated chamber slides  to achieve ∼30% confluency. Trophozoite-infected RBCs were magnetically purified and adjusted to 3–5% parasitemia  and 0.5% hematocrit using uninfected RBCs in binding medium . Parasitized RBC suspensions were added to MVEC-coated chamber slides (200 μl/well) and incubated on an orbital shaker (100 rpm) for 1 h at room temperature. Whenever possible, each parasite isolate was tested for binding in duplicate wells on the same slide. After the parasitized RBC suspensions were removed from each well, the gasket was detached from the slide. The slide was then washed by dipping horizontally in BM four times, fixed in 2% glutaraldehyde overnight at room temperature, and stained in 10% Giemsa for 30 min. The number of parasitized RBCs bound to ∼350 MVECs in each well was counted, and data expressed as number of parasitized RBCs per MVEC. Duplicate comparisons were made whenever sufficient numbers of parasites were available. Seventy-six percent (25/33) of comparisons were performed in duplicate, 50% of which had a ratio of replicate 1∶2 between 0.80 and 1.3 (range 0.5–2.5).An overdispersed Poisson regression was used to model the effect of Hb type (HbAS and HbAC) and age (≤5 and >5 years) on the binding of parasitized RBCs to MVECs, controlling for any ‘slide’ and ‘location within slide’ effects in our data set and for any multiple comparisons done with an individual parasite isolate. Analyses were done in Proc GLMMIX in SAS Version 9.2.ex vivo to the trophozoite stage, magnetically purified, and inoculated into the same wildtype donor RBCs  in an area of intense seasonal transmission for 3 years, we were able to make only 16 comparisons between HbAS and HbAA samples, and only 15 comparisons between HbAC and HbAA samples. The number of comparisons was limited by several factors. First, high numbers of parasites were required to conduct the ex-vivo cytoadherence assay before parasites switched PfEMP1 variants . Second, the assay required that parasites from HbAS (or HbAC) children mature synchronously in culture with those from HbAA children, which did not always occur. Third, HbAS conferred significant protection against malaria in our cohort (unpublished data) and so fewer parasite isolates were available from HbAS children. Additional factors limited data analysis. For example, more comparisons would have enabled us to include age as a continuous variable, providing finer resolution of age effects on cytoadherence. Also, 42% of comparisons between HbAS (or HbAC) and HbAA samples may have been confounded by co-inherited RBC polymorphisms  (We hypothesized that iciency) Table 1,in vivo to modulate the expressed var gene repertoire of parasite populations. Second, if a subset of high-avidity PfEMP1 variants are, indeed, being selected in HbAS children, they might offer an opportunity to identify “virulent” PfEMP1 variants and thus aid the discovery of putative “pathogenicity motifs”  that confer high-avidity binding phenotypes. While such motifs may only cause uncomplicated malaria in older children with HbAS, for example, they may be responsible for causing severe malaria in younger HbAA children who lack both immune- and hemoglobinopathy-mediated resistance to this disease. Such an approach would complement efforts to characterize PfEMP1 variants expressed in children with severe malaria. Since parasites from these children show increased binding to CD36 and ICAM-1, the PfEMP1 variants causing uncomplicated malaria in HbAS children might share features with those causing severe malaria in other children.It remains unclear whether our results reflect the limitations described above or to an actual lack of difference in binding phenotypes for parasites from HbAA and HbAS children – especially since parasites from HbAS children trended towards increased binding to MVECs, but failed to reach significance. However, the lack of statistical significance in both sets of comparisons suggests that any cytoadherence-dependent mechanism is likely working in concert with other factors  to prevent the development of uncomplicated and severe malaria syndromes. Additional studies that are powered to address some of these limitations are therefore warranted since the results from such studies would have two significant implications. First, they would provide insight into whether abnormal PfEMP1 display is working in vivo. Testing the binding of parasites from HbAS, HbAC, and HbAA children with malaria to individual receptors  as well as to MVECs derived from brain or lung may help to further clarify the binding phenotypes of these parasites. Furthermore, identification of “virulent” PfEMP1 domains associated with increased binding to MVECs may inform the rational design of PfEMP1-based vaccines against severe malaria.The results of our study suggest that our particular binding assay detected no clear differences between the cytoadherence of parasites from HbAA, HbAS, and HbAC children to MVECs. Although further studies that are adequately powered to account for age, severity of malaria episode, and co-inherited RBC polymorphisms are needed, our initial findings provide useful insights into the relationship between Hb types and PfEMP1 variants


Plasmodium species while allowing species differentiation would be highly valuable for epidemiology and vaccine studies in areas of mixed infections and identification of malaria-exposed blood donors in non-endemic countries. Here we report a highly sensitive, multiplex ELISA based on recombinant proteins from Plasmodium falciparum, P. vivax and P. malariae malaria for pan-species and species-differentiating detection of antibodies in malaria-positive reference samples and in samples from individuals living in a malaria endemic region in Ghana, Africa.Multiplex ELISA that detect antibodies against more than one falciparum: CSP, AMA-1, LSA-1 and MSP119, P. vivax: AMA-1 and MSP119 and P. malariae: MSP119) were covalently coupled to carboxylated magnetic beads. The dilutions of human plasma/serum were incubated with 3000-5000 antigen-conjugated beads in 96-well plate. Following incubation with a biotin-labeled human anti-IgG conjugate, a streptavidin-PE conjugated fluorescent substrate was added and the plates were read on Bio-Rad BioPlex 200 reader. The reader was set to read a minimum of 50 beads with identical unique detection signal, the results were expressed as median-fluorescent intensity and cut-off titers were established using a pool of normal human serum samples from the US blood donors.Multiplex ELISA was developed utilizing the Luminex xMAP technology that allows the simultaneous detection of antibodies of different specificities that react with antigenic epitopes on multiple beads (microspheres) of different dye intensity. Seven recombinant Plasmodium antigens (P. P. falciparum, P. vivax and P. malariae infected patients. The inclusion of multiple antigens in the multiplex assay makes the test more sensitive than the conventional plate ELISA. The assay was capable to detect differential antibody reactivity to seven Plasmodium antigens in serum samples from 75 adults from malaria endemic area in Ghana who had no demonstrable parasitemia by microscopy. The assay also successfully distinguished between the mixed P. falciparum and P. malariae infections in imported malaria samples obtained in United States.Multiplex ELISA detected 100% of the confirmed malaria reference samples belonging to P. falciparum, P. vivax and P. malariae in a single test format. This assay is being further improved to incorporate the fourth human Plasmodium - P. ovale. We think that this test may be of high value in epidemiological surveys to determine species-specific malaria exposure in areas of mixed infections and vaccine efficacy studies.We have developed a highly sensitive multiplex ELISA that detects the antibodies specific to