Malaria and HIV infections during pregnancy can individually or jointly unleash or confound pregnancy outcomes. Two of the probable outcomes are fetal anemia and cord blood malaria parasitemia. We determined clinical and demographic factors associated with fetal anemia and cord blood malaria parasitemia in newborns of HIV-positive women from two districts in Ghana.P. falciparum in cord blood at delivery) were 57.3% and 24.4% respectively. Factors found to be associated with fetal anemia were maternal malaria parasitemia and maternal anemia. Infant cord hemoglobin status at delivery was positively and significantly associated with maternal hemoglobin and gestational age whilst female gender of infant was negatively associated with cord hemoglobin status. Maternal malaria parasitemia status at recruitment and female gender of infant were positively associated with infant cord malaria parasitemia status.We enrolled 1,154 antenatal attendees (443 HIV-positive and 711 HIV-negative) of which 66% were prospectively followed up at delivery. Maternal malaria parasitemia, and anemia rates among HIV+ participants at enrolment were 20.3% and 78.7% respectively, and 12.8% and 51.6% among HIV- participants. Multivariate linear and logistic regression models were used to study associations. Prevalence of fetal anemia (cord hemoglobin level < 12.5 g/dL) and cord parasitemia (presence of Our data show that newborns of women infected with HIV and/or malaria are at increased risk of anemia and also cord blood malaria parasitemia. Prevention of malaria infection during pregnancy may reduce the incidence of both adverse perinatal outcomes. Malaria in pregnancy may be second to only HIV in terms of mediation of adverse maternal and perinatal outcomes ,2. TheseThere is a general consensus that HIV-uninfected or immunocompetent HIV-infected women with relatively good levels of CD4+ T cells (> 350 cells/μl of blood) can mount protective immune responses when exposed to malaria infection, and this can limit malaria and other infections ,7.Malaria infection (at low density) has been shown to induce and sustain the production of the protective cytokine Interferon gamma (IFN-γ), facilitate protective immune factors such as Leukocyte Inhibitory Factor (LIF), all of which, can reduce HIV-1 replication . Women wIt has been hypothesized that cord blood may become infected with malarial parasites through maternal transfusion into fetal circulation either at the time of delivery, during pregnancy, direct penetration through the chorionic villi, and or premature separation of the placenta . With reet al. . This indicates that the anemic status of mothers have some influence on that of their infants at delivery. These observations are consistent with studies that found high prevalence of fetal anemia in sub-Saharan African countries particularly in malaria-endemic regions [ – 0.55].et al., . It is logical to suppose that gestational age rather has an influence on fetal cord hemoglobin as development of red blood cells in the infant is progressive over the gestation period. This result is supported by the observation that fetal hematocrit increases with gestational age . This co]. It is In the last few weeks of pregnancy (about 32 weeks) matured organs including the kidneys and bone marrow take over the process of erythropoiesis thereby predisposing premature infants whose organs may not be fully matured to fetal anemia .Fetal cord hemoglobin status was negatively associated with female gender of infant [−1.05 ]  before delivery. It is, however, worth noting that in terms of age distribution, gestation length at first antenatal visit, occupation, and socio-economic stratification, those women on whom follow up data were available did not differ significantly from those who were lost to follow-up. Also speculating that the baseline clinical outcomes of this group of women on whom data at delivery were available might differ from those who did not come back to deliver at the study hospitals a similar comparison was made. This analysis again showed that, with the exception of severe anemia at recruitment, the two groups of study participants were comparable (data not shown). On this premise, we proceed to discuss the study findings with confidence that the women on whom the main analysis were based, to a large extent represent the women enrolled into the study.We, however, acknowledge though that, if an intention to treat analysis had been done, possibly different conclusions would have been made. Nevertheless, this approach, which works, on the assumption that none of those lost to follow-up suffered the adverse outcomes of interest can open the door to a misleading presentation of study results. It is also worthy of note that, the other alternative strategies available in dealing with this problem by imputing outcomes to those lost to follow-up, in general, all make unverifiable assumptions that may introduce bias in the estimates of treatment effect. We also acknowledge that several variables including timing of cord clamping could affect the fetal hemoglobin levels. Time of cord clamping was not documented in our study.Our data show that newborns of women infected with HIV, and/or malaria are at increased risk of fetal anemia and also cord blood parasitemia. Fetal cord hemoglobin levels were positively and significantly associated with gestational age and with maternal anemia. This may be uncovering an influence of maternal anemia and the anemic status of infants at delivery. The high level of maternal and infant anemia observed in this study could be attributable to the synergistic effect of HIV and malaria co-infection. Prevention of malaria infection during pregnancy may reduce the incidence of these adverse perinatal outcomes among HIV-positive women.In the settings where both malaria and HIV are co-endemic, routine screening of pregnant women for both malaria and HIV at first antenatal visits and intensification of the education of women at antenatal clinics on adverse perinatal outcomes attributable to malaria in pregnancy may be useful remedying actions. Also, deployment of the various means of preventing malaria, and anemia such as the use of insecticide treated nets and the use of intermittent preventive treatment (IPT) could equip women with self-protection tools. Finally, the promotion and encouragement of diets rich in iron and folic acid may reduce the incidence of anemia among this group of women and ultimately among newborns.The authors declare that they have no competing interest.AKL, WKA, IAQ, JT conceived, designed, implemented the research. AKL, FG, YA, IS, BN, JA, ASL contributed to the data analysis, and drafted different components of the manuscript. All the other authors contributed equally in manuscript proofreading and finalization. All authors read and approved the final manuscript.


Severe malaria is a leading cause of childhood mortality in Africa. However, at presentation, it is difficult to predict which children with severe malaria are at greatest risk of death. Dysregulated host inflammatory responses and endothelial activation play central roles in severe malaria pathogenesis. We hypothesized that biomarkers of these processes would accurately predict outcome among children with severe malaria.Plasma was obtained from children with uncomplicated malaria (n = 53), cerebral malaria (n = 44) and severe malarial anemia (n = 59) at time of presentation to hospital in Kampala, Uganda. Levels of angiopoietin-2, von Willebrand Factor (vWF), vWF propeptide, soluble P-selectin, soluble intercellular adhesion molecule-1 (ICAM-1), soluble endoglin, soluble FMS-like tyrosine kinase-1 (Flt-1), soluble Tie-2, C-reactive protein, procalcitonin, 10 kDa interferon gamma-induced protein (IP-10), and soluble triggering receptor expressed on myeloid cells-1 (TREM-1) were determined by ELISA. Receiver operating characteristic (ROC) curve analysis was used to assess predictive accuracy of individual biomarkers. Six biomarkers  discriminated well between children who survived severe malaria infection and those who subsequently died (area under ROC curve>0.7). Combinational approaches were applied in an attempt to improve accuracy. A biomarker score was developed based on dichotomization and summation of the six biomarkers, resulting in 95.7% (95% CI: 78.1–99.9) sensitivity and 88.8% (79.7–94.7) specificity for predicting death. Similar predictive accuracy was achieved with models comprised of 3 biomarkers. Classification tree analysis generated a 3-marker model with 100% sensitivity and 92.5% specificity .We identified novel host biomarkers of pediatric severe and fatal malaria (soluble TREM-1 and soluble Flt-1) and generated simple biomarker combinations that accurately predicted death in an African pediatric population. While requiring validation in further studies, these results suggest the utility of combinatorial biomarker strategies as prognostic tests for severe malaria. Plasmodium falciparum malaria causes almost one million deaths annually, mostly among young children in sub-Saharan Africa Investigations into malaria pathogenesis have implicated host pathways in disease progression. In particular, dysregulated inflammatory responses and endothelial activation are thought to be central processes in severe malaria pathogenesis Excessive pro-inflammatory responses to infection are observed in both CM and SMA Dysregulated inflammation is thought to promote CM in part through endothelial activation in the brain. Pro-inflammatory cytokines upregulate cell adhesion receptors  that mediate sequestration of parasitized erythrocytes in brain microvasculature, leading to vessel occlusion In this study, we examined plasma biomarkers of inflammation and endothelial activation in children presenting to hospital with malaria. We determined which markers were elevated in severe disease compared to uncomplicated malaria (UM), and which markers discriminated between children who survived severe malaria infection and those who subsequently died. Furthermore, we identified combinations of biomarkers from these two host pathways that accurately predicted mortality among children with severe malaria.Ethical approval for the study was obtained from the Mulago Hospital Research Ethics Committee, Makerere University Faculty of Medicine Research Ethics Committee, Uganda National Council for Science & Technology, and the University Health Network. Written informed consent was obtained from parents/guardians before enrollment.P. falciparum infection . Children were excluded if they had sickle cell trait/disease, HIV co-infection, or severe malnutrition. Clinical and demographic data were collected upon enrollment, and venous blood samples were collected for routine measurement of hemoglobin and platelet count, and for plasma banking. Thin blood smears were obtained at presentation for determination of parasitemia, which is reported as the arithmetic mean of two independent readings by expert microscopists. Treatment was in accordance with Ugandan national guidelines: artemether/lumefantrine was administered to children with uncomplicated malaria, and parenteral quinine was used in severe malaria cases This retrospective case-control study was nested within a larger study conducted at Mulago Hospital in Kampala, Uganda between October 2007 and October 2009. Mulago Hospital is a national referral hospital that serves Kampala and surrounding districts. Malaria transmission in this region and the patient population at Mulago Hospital have been previously described P. falciparum parasitemia , or repeated (>3) seizures witnessed within a 24 h period, in the absence of hypoglycaemia (<40 mg/dL or 2.2 mM) or any known alternative neurologic abnormalities). SMA was defined as hematocrit<15% or hemoglobin<5.0 g/dL in the presence of asexual parasitemia.For biomarker analysis, a sub-group (n = 156) of UM outpatients, CM inpatients, and SMA inpatients in roughly equal numbers was selected from the larger study based on availability of an adequate volume of previously unthawed plasma. CM was defined as an unrousable coma (not attributable to any other cause) in a child with asexual 2SO4, and read at 450 nm. Background signal was determined from blank wells included on each plate (assay buffer added instead of sample), and background optical density was subtracted from all samples and standards prior to analysis. Samples with optical densities below the lowest detectable standard were assigned the value of that standard.Plasma (sodium citrate anticoagulant) was stored at −20°C prior to testing. ELISAs were used to quantify plasma biomarker levels and were performed blinded to all associated clinical data. The following markers were assayed (dilution factor indicated in parentheses): Ang-2 (1∶5), CRP , sTREM-1 (neat), s-endoglin (1∶25), IP-10 (1∶2), sFlt-1 (1∶6), sICAM-1 (1∶1000), sP-selectin (1∶50), sTie-2 , PCT , and vWF propeptide . ELISAs were performed according to the manufacturers' instructions, with the following changes: assays were performed in a volume of 50 µL/well; plasma samples were incubated overnight at 4°C; and ELISAs were developed using Extravidin®-Alkaline Phosphatase  followed by addition of p-Nitrophenyl phosphate substrate (Sigma) and optical density readings at 405 nm. s-endoglin and sP-selectin were only measured in samples collected during the first year of the study (n = 101). For vWF, plates were coated with anti-human vWF antibody , incubated with samples (1∶500 dilution) and serial dilutions of recombinant vWF (American Diagnostica), then incubated with horseradish peroxidase-conjugated anti-human vWF . Assays were developed with tetramethylbenzidine, stopped with HGraphPad Prism v4, SPSS v18, and MedCalc software were used for analysis. For clinical and demographic variables, differences between groups were assessed using the Chi-square test  or the Kruskal-Wallis test with Dunn's multiple comparison post-hoc tests (continuous variables). The Mann-Whitney U test was used to compare biomarker levels between groups, and p values were corrected for multiple comparisons using Holm's correction. Receiver operating characteristic curves were generated using the non-parametric method of Delong et. al Children presenting to Mulago Hospital in Kampala, Uganda with UM (n = 53), CM (n = 44), and SMA (n = 59) were included in the study. Six children had concurrent CM and SMA and were categorized as “CM”, and five children with SMA exhibited decreased consciousness but did not meet study criteria for CM. Plasma samples obtained at presentation were assayed for biomarkers of endothelial activation and inflammation . sICAM-1To evaluate the prognostic utility of these plasma biomarkers, we compared admission levels between children with severe malaria who survived infection and those who subsequently died. After correction for multiple comparisons, admission levels of Ang-2 were significantly increased in CM fatalities compared to survivors ; p<0.05,The biomarkers that reached significance in the SMA group but not the CM group after correction for multiple comparisons  were significant or trending towards significance in the CM group before the correction was applied. This suggests that the apparent differences between syndromes may have been due to low statistical power and therefore we combined all severe malaria patients for further analysis. This strategy also avoids the problem of classifying mixed clinical phenotypes, as occurred in the present study population. Characteristics of survivors and fatalities were similar , althougTo assess how well these biomarkers discriminated between survivors and fatalities, we generated receiver operating characteristic (ROC) curves and determined area under the curve (AUC) . Ang-2, We used the Youden index to obtain a cut-point for each biomarker, and evaluated clinical performance measures for these dichotomized biomarkers . sTREM-1We hypothesized that combining biomarkers would improve predictive accuracy. The modest number of deaths in the study precluded multivariable logistic regression analysis with more than 2–3 independent variables Biomarker score was highly positively correlated with risk of death , Model 1ROC curve analysis and cut-point determination were performed as above for the biomarker score. The AUC was 0.96 (0.90–0.99) , and we A score involving fewer biomarkers might be expected to improve practicality and facilitate potential translation to a clinical application. Using the same scoring scheme, 2-marker combinations performed poorly (data not shown). However, specific 3-marker combinations yielded sensitivity>90% and specificity>80% .To explore another combinatorial strategy, we used classification tree analysis, which selects and organizes independent variables into a decision tree that optimally predicts the dependent measure. Initially, a model based on IP-10 and sTREM-1 was generated with 43.5% sensitivity and 100% specificity for predicting mortality (data not shown). Since high sensitivity would be a crucial feature of a prognostic test for severe malaria, we repeated the analysis assigning the cost of misclassifying a death as a survivor as 10 times greater than the cost of misclassifying a survivor as a death. A model based on IP-10, Ang-2, and sICAM-1 was generated , with 10Combinations of prognostic biomarkers, particularly if drawn from distinct pathobiological pathways, have been found to improve predictive accuracy We found that plasma sTREM-1 levels reflected disease severity in children with severe malaria. The TREM-1 receptor is expressed on the cell membrane of monocytes and neutrophils and mediates pro-inflammatory responses sFlt-1 is generated by alternative splicing of VEGF receptor-1 mRNA and antagonizes the pro-inflammatory and pro-angiogenic effects of VEGF. Our observation of increased sFlt-1 in severe malaria parallels findings in sepsis patients There were some discrepancies between our data and previous studies of biomarkers in pediatric severe malaria. s-endoglin was found to be increased in Gabonese children with severe malaria compared to UM As previously described Regardless of whether these biomarkers mediate or simply reflect pathology, combinations of biomarkers accurately predicted mortality among children with severe malaria in our sample. Notably, some biomarker combinations showed excellent sensitivity, ensuring that the majority of children at high risk of death would be identified. While an effective adjunctive therapy for severe malaria remains elusive, prognostication could allow triage of patients for closer monitoring or intensive care resources, as available. Such a test may also assist in risk stratification and patient selection for clinical trials of adjunctive therapies, which are ongoing Previous studies have developed clinical scores to prognosticate outcome in pediatric severe malaria The limitations to our study include a small sample size and the use of non-consecutive samples, which may have introduced a selection bias. Co-infections with pathogens other than HIV were not assessed, and thus it is unclear how they may have affected biomarker levels. Biomarker combinations that accurately predicted mortality require validation in larger prospective studies with adjustment for potential demographic and clinical confounders and head-to-head comparison with prognostic clinical scores. Furthermore, these combinations require validation across different ethnicities and malaria endemicities, as well as in children with non-CM/non-SMA severe malaria syndromes. Nevertheless, this study identified novel biomarkers in African children, who are at the greatest risk of malaria mortality, and specifically in SMA, for which few informative biomarkers have been described. We provide proof-of-concept that combining as few as 3 biomarkers using simple schemes may be able to accurately predict outcome in severe malaria infection.


In tropical areas, the predominant cause of fever has historically been malaria. However by 2011, among febrile patients in northwestern Ecuador, dengue was identified in 42% and malaria in none. This finding suggests a transition in the cause of fever from malaria to other illnesses, such as dengue. Aedes aegypti mosquitos  was introduced into Esmeraldas in 1990 and was the only documented serotype circulating until 2004, when DENV-4 was identified. DENV-4 was thought to arrive in Esmeraldas from Colombia and subsequently spread into the rest of Ecuador , where patients who suspect that they have malaria often go, or the Hospital Civil de Borbón (HCB), the main hospital in the region. We provide data from Borbón, a rural community in northwestern coastal Ecuador, as a case study of this etiologic transition of fever from malaria to other illnesses, such as dengue. In addition, we provide serotype-specific data that describe the directional flow of dengue at the regional and national levels.At HCB, as part of the routine workup for febrile patients, serum samples were collected, stored at −20°C, and transported in liquid nitrogen to Quito for PCR. Hospital personnel also tested these samples by using a dengue IgM/IgG-capture ELISA with high specificity and sensitivity . At the SNEM laboratory, 2–4 drops of whole blood were collected on filter paper  from persons spontaneously seeking or referred for malaria diagnosis. Filter papers were left to dry overnight, stored at 0°C in a zipper bag, and transported to Quito for analysis. DENV was amplified from total RNA from blood spots and serum as described  were collected from febrile patients. Of these 36 hospital patients, 10 were from Borbón and 26 were from the surrounding communities; patients were 2–74 years of age (median 19 years), and fever duration was 3–20 days (median 6 days). Six (17%) of the 36 serum samples and 7 (17%) of the 41 blood spots were positive for dengue by PCR; whereas 10 (29%) of the 34 serum samples tested by ELISA were positive for dengue IgM . The agePlasmodium spp., traditionally highly prevalent in this area, to reemerging pathogens, such as DENV. Other possible fever-causing agents for which we did not test include Leptospira spp., Rickettsia spp., Brucella spp., and viral agents of encephalitis (This study provides evidence of a transition of febrile disease etiology from


Plasmodium falciparum Erythrocyte Binding Antigen-175 (EBA-175) is an antigen considered to be one of the leading malaria vaccine candidates. EBA-175 mediates sialic acid-dependent binding to glycophorin A on the erythrocytes playing a crucial role during invasion of the P. falciparum in the host cell. Dimorphic allele segments, termed C-fragment and F-fragment, have been found in high endemicity malaria areas and associations between the dimorphism and severe malaria have been described. In this study, the genetic dimorphism of EBA-175 was evaluated in P. falciparum field isolates from Brazilian malaria endemic area.The The study was carried out in rural villages situated near Porto Velho, Rondonia State in the Brazilian Amazon in three time points between 1993 and 2008. The allelic dimorphism of the EBA-175 was analysed by Nested PCR.The classical allelic dimorphism of the EBA-175 was identified in the studied area. Overall, C-fragment was amplified in a higher frequency than F-fragment. The same was observed in the three time points where C-fragment was observed in a higher frequency than F-fragment. Single infections (one fragment amplified) were more frequent than mixed infection (two fragments amplified).These findings confirm the dimorphism of EBA175, since only the two types of fragments were amplified, C-fragment and F-fragment. Also, the results show the remarkable predominance of CAMP allele in the studied area. The comparative analysis in three time points indicates that the allelic dimorphism of the EBA-175 is stable over time. Plasmodium responsible for human infection, Plasmodium falciparum is the most virulent species, mainly by wide spectrum of clinical complications. Considering the number of malaria cases and the increasing resistance of the Plasmodium to anti-malarial drugs [Anopheles spp. to insecticides [P. falciparum, the species responsible for severe malaria and mortality. One approach to developing a malaria vaccine is focused on preventing the interaction between merozoite surface ligands and the erythrocyte receptors, a short period in which the parasite is vulnerable to attack by the immune system [Malaria is an important parasitic disease responsible for around one million deaths per year, especially in developing countries . Among tal drugs -5 and thcticides ,7 the coe system .P. falciparum merozoites uses a 175-kDa sialic acid-binding protein ligand to invade the host erythrocyte and this protein is known as erythrocyte binding antigen 175 (EBA-175) [eba-175 gene is located on chromosome 7 and comprises four exons and seven regions, named I to VII. The region III is located in the central part of the gene and studies have shown a highly dimorphic segment in this region. This dimorphism is characterized by the insertion of a segment of 423 base pairs (bp) in strain FCR3 (F-fragment) or a segment of 342 bp in strain CAMP (C-fragment). These two variants are conserved among strains of P. falciparum, and considering that the merozoites are haploid and eba-175 is a single copy gene, either one or the other segment is present in a uniclonal infection [One of the major antigens of EBA-175) ,10. EBA-EBA-175) . The ebanfection -14. The nfection . Howevernfection ,16.P. falciparum isolates from Rondonia State, a Brazilian malaria endemic area.Several studies performed in malaria hyperendemic areas in Africa have shown the influence of this dimorphism, more precisely EBA175 allele distributions, on clinical disease and outcome ,13,14. TThe study was carried out in rural villages situated near Porto Velho, the capital of Rondonia State, malaria endemic region in the Brazilian Amazon Figure . In thisP. falciparum infected individuals: 101 in time point-1, 57 in time point-2 and 30 in time point-3. Samples from the three time points were used in order to evaluate whether the allelic dimorphism of the EBA-175 in Brazil was present and if this dimorphism is temporally stable in the same area. All P. falciparum malaria patients enrolled in this study presented uncomplicated malaria symptoms; positive thick blood smear for P. falciparum and were not under chemoprophylaxis nor took anti-malarial drugs (self-treatment). Blood collection was performed on the day of diagnosis before malaria treatment and the patients sought health care at Brazilian health services 3.6 ± 1.8 days after onset of symptoms. After blood sample collection, the patients were immediately treated according to Brazilian Ministry of Health standards for malaria therapy. The study was reviewed and approved by the Fundação Oswaldo Cruz Ethical Committee.Written informed consent was obtained from all donors and venous blood samples were taken from P. falciparum in the thick and thin blood smears were enrolled in the study. The P. falciparum parasite density was determined by counting parasites in a predetermined number of white blood cells in thick blood films, and the number of blood parasites per milliliter was calculated [2).Thin and thick blood smears were examined for identification of malaria parasite by a technician experienced in malaria diagnosis from Brazilian Malaria Health Services and from the Laboratory of Malaria Research (Fiocruz) which is a reference in malaria diagnosis from Brazilian Ministry of Health. Thick blood smears from all subjects were stained with Giemsa, and a total of 200 microscopic fields were examined under 1,000-fold magnification. Thin blood smears of positive samples were examined for species identification. Only patients infected with lculated . The freeba-175 gene, the DNA present in 1 mL of cryopreserved blood was extracted using the QIAamp DNA blood midi kit (QIAgen) as described by the manufacturer and stored at -20°C until amplification. The region III of the eba-175 gene was amplified in a Nested PCR method as described elsewhere [® DNA polymerase , 5 μL of 10× PCR-Buffer (Applied Biosystem) and 2.5 mM of MgCl2 (Applied Biosystem). The Nested PCR reactions were carried out using a GeneAmp® PCR System 9700 (Applied Biosystem) for thirty cycles . Fifteen μL of PCR-product were loaded onto a 2% agarose gel (LGC Biotecnologia Ltda) in 1× TAE buffer  in the presence of ethidium bromide (0.5 μg/mL).In order to amplify the region III of the lsewhere  using th®  data bank software. Statistica  and Epi-Info 6  statistical software programs were used for data analysis. The Student's t-test was used to analyse the differences in mean values and the chi-square test was used to analyse the difference in prevalence of allelic frequencies.The data were stored in the Fox-plusP. falciparum samples were collected from malaria patients in time point-1 (n = 101), time point-2 (n = 57), and time point-3 (n = 30). The composition of individuals in the three time points was similar regarding gender. Males were predominant in three time points, 69%, 83% and 77% in the time points-1, -2 and -3, respectively. The average age was also similar among individuals in time point-1 (31 ± 13 years old), time point-2 (29 ± 11 years old) and time point-3 (31 ± 10 years old). As expected, the time of residence of individuals in the endemic regions was higher in the time point-3 (28 ± 12) than in the time point-1 (12 ± 9) and time point-2 (19 ± 9) . The means parasitaemia at the time of blood collection were 7751 ± 8431, 2218 ± 738 and 3587 ± 3769 parasites/μL in the time points-1, -2 and -3, respectively. In the time point-1 the mean parasitemia was higher than in time points-2 and -3 .Throughout the study period, a total of 188 P. falciparum wild isolates were, respectively, 83.2% and 16.8%. The predominance of the C-fragment was evident in the P. falciparum samples from studied area .The two types of fragments were identified in the studied area by Nested PCR, one of 714 bp, corresponding to the C-fragment, and the other one of 795 bp, corresponding to the F-fragment Figure . The freP. falciparum isolates presented only one amplified fragment (single infection), while both fragments were observed in only 1.6% (3/188) of the isolates representing mixed infections .The vast majority  of the P. falciparum samples from studied area in the three time points .As can be seen in Table P. falciparum isolates from Brazilian malaria endemic area, Rondonia State, in three different time points  and F-fragment (795 bp), were identified in the studied area. Overall, C-fragment (83.2%) was observed in a higher frequency than F-fragment (16.8%). This data differ from results reported by three independent research groups in high endemicity malaria areas of Ghana, Gabon and Burkina Faso where F-fragment was observed in a higher frequency than C-fragment [Plasmodium populations in infected studied individuals and/or genetic background of the human studied population [The aim of this study was to investigate the allelic dimorphism of the EBA-175 in fragment ,13,19. Ipulation ,22. AlsoThe differences observed in the distribution of C- and F-fragments among geographical regions could be explained by random shifts in parasite allele frequencies due to genetic drift in genetically isolated populations . In thisP. falciparum genetic diversity, the individuals have a higher probability of being infected simultaneously with more than two clones of the parasite, which has been called the multiclonal [Plasmodium spp. in the mosquito and thus increase the genetic diversity [P. falciparum isolates from studied population was evaluated. In the present work, a low frequency of mixed infection  was found. These results are similar to a study with the P126 protein that reveals low frequency of mixed infection in Brazilian isolates from the same area [In areas of high ticlonal ,25 or miiversity ,27, the ame area . The thrame area ,30. AlteP. falciparum isolates from Rondonia State in three time points over 15 years suggest that the frequencies of the two allelic forms remains stable over time. These data supports the hypothesis of a biologically important role for EBA-175 in parasite development and/or survival and highlight the importance of evaluating the distribution of EBA-175 allelic forms in different geographical areas.In conclusion, in the studied area, C-fragment is more frequent than F-fragment. The comparative analysis of the allelic dimorphism of the EBA-175 in The authors declare that they have no competing interests.DSPS participated in the design of the study; carried out the molecular study; performed the statistical analysis and drafted the manuscript; DMB and LRPR conceived of the study, and participated in its design and coordination and helped to draft the manuscript. JCLJ, FS, CTDR and JOF helped in the design of the study and reviewed the manuscript. All authors have read and approved the final manuscript.


Plasmodium parasites. The determinants of gametocyte carriage have been studied extensively in endemic areas, but have rarely been explored in travellers with malaria. The incidence of gametocytaemia, and factors associated with gametocyte emergence in adult travellers with Plasmodium falciparum malaria was investigated at the Hospital for Tropical Diseases in London.Gametocytes are the sexual stage of P. falciparum malaria between January 2001 and December 2011 were extracted from a prospective database. These data were supplemented by manual searches of laboratory records and patient case notes.Clinical, parasitological and demographic data for all patients presenting with P. falciparum malaria were identified. Four hundred and sixty five (60%) were born in a country where malaria is endemic. Patients presented to hospital a median of four days into their illness. The median maximum parasite count was 0.4%. One hundred and ninety six patients (25%) had gametocytes; 94 (12%) on admission, and 102 (13%) developing during treatment. Gametocytaemia on admission was associated with anaemia and a lower maximum parasitaemia. Patients with gametocytes at presentation were less likely to have thrombocytopenia or severe malaria. Patients who developed gametocytes during treatment were more likely to have had parasitaemia of long duration, a high maximum parasitaemia and to have had severe malaria. There was no apparent association between the appearance of gametocytes and treatment regimen.Seven hundred and seventy three adult patients with laboratory-confirmed P. falciparum is associated with factors similar to those reported among populations in endemic areas. These data suggest that acquired immunity to malaria is not the only determinant of patterns of gametocyte carriage among patients with the disease.The development of gametocytaemia in travellers with  Plasmodium falciparum, gametocytes arise seven to 15 days after the initial patent parasitaemia and last for a mean of 6.4 (range 2.5–22) days in the circulation, longer than the typical duration of asexual parasitaemia [Gametocytes are the sexual stage of malaria parasites. The generation of male and female gametes is necessary for transmission to the mosquito vector where sexual recombination and generation of new haploid progeny takes place. In acute infection with .4 range .5–22 daysitaemia , the dursitaemia .P. falciparum. Travellers represent an interesting cohort for two reasons. First, they usually present early in the natural history of their disease, and second, they may have little immunity to malaria. This provides an opportunity to analyse the effect of other variables, such as the duration of infection and the impact of disease severity, on the likelihood with which gametocytes are seen. In this study, factors associated with gametocytaemia were assessed among a population of adult travellers with P. falciparum malaria at the Hospital for Tropical Diseases, London, UK.Few data have been published on gametocyte carriage among travellers with P. falciparum between the beginning of January 2001 and end of December 2011 were included. The diagnosis of malaria was made by the UK’s Parasitology Reference Laboratory using standard thick and thin films which were, for those admitted, repeated daily until asexual parasites were no longer seen. Severe malaria was defined according to the unit’s guidelines: parasitaemia >2% and/or the presence of complications [2 distribution. Continuous variables were compared across binary categories using the Wilcoxon rank-sum test. Multivariate logistic regression was performed on those (binary) variables found significantly associated with gametocyte carriage in the univariate tests. All statistical analyses were performed in STATA . The study was reviewed and approved by the Audit and Research Committee at HTD who stated that, as this was a retrospective case note review, further formal ethical approval was not required.The Hospital for Tropical Diseases is a tertiary referral hospital in London. Patients may present directly, by general practitioner referral or as a transfer from another hospital. Demographic details of all patients admitted to the hospital with falciparum malaria are prospectively collected into a database. Patients treated as outpatients were identified by reviewing laboratory records. Further data were collected from laboratory and case note records. All patients diagnosed with ications . ComplicP. falciparum malaria were identified. Forty-nine of these were under the age of 18 years and were excluded as their biological response to malaria differs from adults. This left a total adult population of 773.Eight hundred and twenty-two patients with laboratory-confirmed Median age was 38 years and 63% were male. Four hundred and sixty five (60%) had been born in a malaria-endemic country and may have had partial immunity to the disease. Two hundred and sixty nine (34.8%) had been born in a country without malaria transmission and were assumed to be malaria-naïve Table . The remRelatively few patients had taken appropriate anti-malarial prophylaxis Table . Median Patients who had gametocytaemia at presentation were more likely to have been ill for longer, to be anaemic and to have a normal platelet count Table . In addiMultivariate logistic regression was performed with five parameters significantly associated with the presence of gametocytes at presentation (in decreasing order of apparent strength of association): anaemia, thrombocytopenia, asexual parasitaemia, duration of illness and severe anaemia Tables  and 3. TThose who developed gametocytes during treatment were more likely to have presented with a higher parasitaemia and to have at least one marker of severe disease compared to those who did not Table . In addiMost patients 643, 83%) were treated with quinine. Artemisinin treatment was postulated to be associated with a lower rate of gametocytaemia development post-treatment. No evidence was found to support this among the 54 patients receiving an artemisinin-containing regimen (p = 0.226). However, this analysis was confounded by the fact that patients treated with artemisinins were more likely to be severe malaria cases  and those with severe malaria were independently more likely to develop gametocytaemia following anti-malarial treatment  and less likely to have thrombocytopenia (associated with acute illness) than those who did not have gametocytes at presentation. The data also suggest that despite the chronicity of their illness, these patients were less unwell; they tended to have a lower parasitaemia and fewer of them had any markers of severe malaria.P. falciparum develops among children who live in highly endemic areas and survive repeated infections [There are two principal explanations for the ability of some patients to tolerate malarial infection for longer without becoming unwell. One is the use of partially effective infrequent anti-malarial chemoprophylaxis, which may attenuate an infection without clearing it. The other is previous exposure to malaria, which may induce acquired immunity. There was no demonstrable association between prophylaxis use (either complete or incomplete) and gametocytaemia at presentation. It is well documented that immunity to fections ,12. It mPatients who have not grown up in an endemic area are usually considered to be malaria-naïve. Intuitively, this group may be considered at greater risk of severe disease and less likely to tolerate malaria infection. However, this group had a similar rate of gametocytaemia at presentation and were no more likely to have markers of severe disease. In this group, factors associated with gametocytaemia  mirrored the findings from studies in endemic areas. In a three-centre study, gametocytaemia at presentation was seen in 35% of cases from Thailand, 37% of cases from The Gambia and 26% of cases from Tanzania . The ratP. falciparum, which indicate that previous infection history is a much less critical factor than it is for immune modulation of asexual parasitaemia [The data indicate that although previous exposure may be important in determining which patients present with gametocytaemia, this is not the full explanation. It seems conceivable that some form of innate immunity may also play a role in modulating the nature of the illness and the likelihood of gametocytes being present early in the clinical disease. A number of factors have been associated with the potential to protect against severe complications of malaria, but contribute towards anaemia, such as sickle cell trait . Howeversitaemia ,12,15.The emergence of circulating gametocytes in patients receiving anti-malarial treatment has been associated with the time taken to clear asexual parasites from the peripheral circulation . The datNo evidence was found that gametocyte carriage among adult malaria patients in the UK was related to previous exposure to malaria. This suggests that acquired immunity alone is not the sole determinant of gametocyte emergence in clinical cases.The authors declare there are no financial relationships with any organisations that might have an interest in the submitted work, nor other relationships or activities that could appear to have influenced the submitted work or its interpretation.Data collection was performed by CHR, MA, EZ, SB, SW and PLC. Analysis was performed by all authors. The manuscript was drafted by CHR, and revised by MA, CJS, and TD. All authors read and approved the final manuscript.


The current target of universal access to long-lasting, insecticide-treated nets (LLIN) is 80% coverage to reduce malaria deaths by 75% by 2015. So far, campaigns have been the main channel for large-scale delivery of LLINs, however the World Health Organization has recommended that equal priority should be given to delivery via routine antenatal care (ANC) and immunization systems (EPI) to target pregnant women and children from birth. These various channels of LLIN delivery are targeted to children of different ages. Since risk of mortality varies with child age and LLIN effectiveness declines with net age, it was hypothesized that the age at which a child receives a new LLIN, and therefore the delivery channel, is important in optimizing the health impact of a net.A simple dynamic mathematical model was developed of delivery and impact of LLINs among children under five years of age and their household members, incorporating data on age-specific malaria death rates, net use by household structure, and net efficacy over time.The presented analysis finds that supplementing a universal mass campaign with extra ANC delivery would achieve a 1.4 times higher mortality reduction than campaign delivery alone, reflecting that children born in the years between campaigns would otherwise have access to old nets or no nets at an age of high risk. The relative advantage of supplementary ANC delivery is still present though smaller if malaria transmission levels are lower or if there is a strong mass effect achieved by mass campaigns.These results indicate that LLIN delivery policies must take into account the age of greatest malaria risk. Emphasis should be placed on supporting routine delivery of LLINs to young children as well as campaigns. Long-lasting, insecticide-treated nets (LLINs) are one of the most efficacious preventive interventions against malaria morbidity and mortality available and formThe RBM strategic plan recommends that the 80% universal coverage target is achieved using a combination of campaigns and continuous channels such as routine antenatal clinics (ANC) and the expanded programme of immunization (EPI) for LLIN delivery, the so-Despite these tremendous efforts, emerging data indicate that use of LLINs is not sustained at high levels over time. For example, in Togo and Sierra Leone, the percentage of children under five sleeping under a net dropped to around 50% a year after mass distribution campaigns, and was only 25-30% 18 months later. In otheNationally representative data on LLIN ownership and utilization used to measure progress towards the RBM targets is collected via standard RBM malaria modules included in Demographic and Health Surveys (DHS), UNICEF Multiple Indicator Cluster Surveys (MICS) and Malaria Indicator Surveys (MIS). However, questions on the source of LLINs being used by household members is a relatively recent addition and very few countries, therefore, have data on the relative contribution of campaigns and specific routine or continuous delivery channels  to LLIN ownership. Thus, aCoverage achieved by different delivery systems will influence the potential impact on mortality that LLIN programmes can have, likewise the well-documented discrepancy between ownership and use. UniversIt is clear from available evidence that campaigns alone are not currently achieving or sustaining the 80% targets for LLIN use,13,14. CA similar modelling approach to the Lives Saved Tool (LiST) model was adopted, where tet al.[The under five-year-old population was modelled in six-month age bands. Three different distributions of age-specific death rates were explored within this group, representing high, medium and low malaria transmission. For the high transmission scenario, age-specific malaria mortality rates averaged across five sites in sub-Saharan Africa were used, where met al.. The ageDemographics in older children and adults are not explicitly modelled but coverage and use of LLINs among this group as a whole is tracked in order to model the potential mass effect in the total population acting upon children under five years (see below).Use of a new LLIN is assumed to reduce malaria mortality by 55%. It is aFour different delivery strategies for LLIN distribution are included in the model: giving an LLIN (i) to pregnant women at ANC clinic attendance; (ii) to infants attending EPI (routine delivery); or distributing LLINs via mass distribution campaigns, involving (iii) children under five only (targeted campaign); or, (iv) the whole population . Each delivery strategy may be implemented alone or in combination. The assumptions associated with each strategy are described as follows.ANC delivers a LLIN to the mother during pregnancy. It is assumed for simplicity that the LLIN is not used until the child is born, i e, a In the model, LLINs delivered via EPI are received by the infant at age six months. With the exception of BCG vaccination, which is scheduled to be administered at birth, EPI visits are scheduled from one to nine months. However, based on a multi-country analysis of actual timing of EPI attendance it is assumed that a high proportion of infants are brought late for their first EPI visit, or do not receive an LLIN until their second EPI visit due to imperfect distribution, giving an average age of six months when an LLIN is received. Once anA targeted campaign delivers one LLIN to each child under five, while a universal campaign delivers two LLINs to each household. Campaigns are carried out every three years.Average LLIN ownership over time was calculated both for children in each six-month age group among the under-fives, and as a total by household, according to the combined coverage of all the delivery strategies described above  was calculated and stratified by the number of children under five living in the household  and the age of the newest net. The probability of a child belonging to a household with a given number of under-five children was set according to the household structure recorded in the Tanzania DHS survey in 2005  reflecting the shift in risk of malaria mortality to older age groups. However, sharing of LLINs among members of the household and mass effect of the intervention may affect these comparisons of routine vs campaign delivery strategies, and these factors are therefore incorporated in the analysis that follows.Routine LLIN delivery at birth predicts more deaths averted among under-fives than a campaign LLIN delivery in high and medium transmission settings FigureA and2B,The effects of household structure on ownership and use of LLINs by under fives in the household were incorporated to generate the full model. The number of malaria deaths averted per 1,000 children under five every six months and the cumulative under-five deaths averted per 1,000 LLINs delivered were calculated for each delivery scenario. All children were included in these calculations whether they received an LLIN or not. In the base case, the universal target of 80% coverage (taken here to be ownership of 1+ LLINs) was assumed for each delivery channel and any potential mass effect on the vector population was ignored Figure. For theIn all malaria transmission settings, the pattern of malaria deaths averted for routine delivery channels shows a gradual increase initially as infants are covered with an LLIN and age into older age groups, then reaches equilibrium once the first infants to receive the intervention are five years old. The level of this equilibrium and the relative impact of LLINs delivered via ANC compared to EPI differ by transmission intensity, reflecting the different age patterns of malaria mortality. For example, in the high transmission setting, 1.47 deaths per 1,000 children could be averted per six months by LLINs delivered through ANC and used from birth, compared to 1.35 by LLINs delivered through EPI at six months of age  FigureA, impactHigher coverage of routine LLIN delivery channels, whether in combination with low or high universal campaign coverage, maintains a greater number of deaths averted between campaigns FigureB.vs 1.4-fold in a higher transmission scenario).Similar patterns were found for the predicted impact of different combinations of delivery channels in areas of medium and low malaria transmission, although total malaria mortality is lower and so the numbers of deaths averted are fewer (results not shown). There were two differences of note. Firstly, in both medium and low transmission scenarios, LLINs delivered through EPI had a greater potential impact than those delivered through ANC, reflecting the greater risk of malaria mortality in older age groups. Secondly, adding ANC delivery to campaign delivery gave a slightly smaller relative gain in low to medium settings although the difference was not great. For example, ANC added to a campaign still increased the number of deaths averted 1.3-fold at low transmission . If considering ‘efficiency’ where the impact on mortality is presented in relation to the number of LLINs delivered, delivery of a very large number of nets will improve mortality outcomes but reduce efficiency since there will be some overlap in coverage. The balance between effectiveness and efficiency presents a dilemma for control programmes and ministries of health. This model predicts the greatest efficiency for delivery of LLINs through ANC or EPI if only one delivery channel is used; however these routine delivery systems alone do not achieve high enough coverage. Maximum effectiveness is achieved by delivering through as many channels as possible. Perhaps the compromise is LLIN delivery through one routine channel, the choice of which will depend upon context in terms of transmission intensity, together with regular campaigns, which would achieve good levels of effectiveness for a medium level of efficiency.Nevertheless, the cost per LLIN delivered for each delivery system combination has to be considered. Provider-side financial cost per treated net delivered through routine ANC at district scale ranged from US$7.64 in Kenya to US$8.Equity should also be considered, and evidence suggests that the same children may be missed by all interventions due to lThe results presented here support the RBM-recommended strategy of a combination of routine and campaign LLIN delivery. However, the focus over the last few years has been on campaigns and the process of ‘catch up’ to progress towards the universal coverage goal. Reasonable results have been shown for a number of routine delivery strategies,8,28,34,This analysis used a simple model, which has the advantage of greater transparency and fewer assumptions about uncertain factors, but also has limitations. For example, the data used to model household structure and its influence on net use is specific to Tanzania and this may show different patterns in other countries with alternative household structures; it would be of interest to explore the effect of altering these parameters on model predictions in future analyses. The model also does not allow for changing immunity profiles as a result of LLIN introduction, which may shift mortality burden to older age groups. This factor was not included because the timescale over which immunity is lost is highly uncertain, and therefore any model dependent on this would be extremely reliant on which assumptions were made. So far there is no evidence that LLINs do shift mortality to older ages-45. Howevs non-users was assumed throughout the under-five cohort, as supported by evidence from an intervention trial[A constant relative risk of malaria mortality among LLIN users on trial. Howeveron trial. If thison trial. Althougon trial, this reon trial,49. TherOne of the arguments for the expansion of LLIN delivery to all age groups rather than only the biologically vulnerable is the potential reduction in transmission and related health burden at the community level (the mass effect), which has been demonstrated by field data,50 and svs older children. We assumed homogeneous conditions across the modelled population, for example regarding transmission intensity and use of nets. To capture a more realistic heterogeneous population, for example to model potential impact of campaigns targeted at particular geographical areas, it would be necessary to vary these factors in order to represent sub-populations of interest.There is some evidence that the proportional reduction in all-cause under-five mortality achieved by insecticide-treated nets is higher at low transmission intensities but thisPrevious mathematical models have examined the effects of LLINs on malaria transmission at a population level, using a full transmission model framework and focusing on reductions in prevalence of infection and effects on the vector population-53; diffMathematical modelling confirms that in order to maximize the number of deaths of children under five that can be averted by LLINs, delivery through routine ANC and/or immunization services is necessary in addition to delivery through campaigns. Regardless of the level of mass effect, average cumulative health impact  was greater with LLINs delivered to an infant through ANC or EPI than with LLINs delivered through campaigns alone. This reflects that when a new LLIN is delivered to a child at their age of greatest risk of malaria mortality, the period of greatest vulnerability in the child coincides with the period of greatest protective efficacy in the net. Strong emphasis should be placed on supporting routine delivery of LLINs and not focusing resources purely on campaigns. A transition from a campaign focus to one balanced with routine service delivery will require a well-coordinated and funded effort between national programmes and their partners to assure well-functioning infrastructure for routine delivery is in place. More lessons need to be collated and capacity strengthened for effective delivery of LLINs through routine channels.The authors declare that they have no competing interests.JL, LSP, JW and KH devised the study design and objectives. LO developed the model. LO, LSP, JL, JW and KH contributed to parameterization, analysis and interpretation. LSP and LO wrote the first draft of the manuscript. All authors read, commented on and approved the final manuscript.Supplementary information on parameter assumptions and data sources.Click here for file


Plasmodium knowlesi is recognized as a common cause of severe and fatal human malaria in Sabah, Malaysia, but is morphologically indistinguishable from and still commonly reported as Plasmodium malariae, despite the paucity of this species in Sabah. Since December 2008 Sabah Department of Health has recommended intravenous artesunate and referral to a general hospital for all severe malaria cases of any species. This paper reviews all malaria deaths in Sabah subsequent to the introduction of these measures. Reporting of malaria deaths in Malaysia is mandatory.The simian parasite Plasmodium species. Demographics, clinical presentations and management of severe malaria caused by each species were compared.Details of reported malaria deaths during 2010-2011 were reviewed to determine the proportion of each Plasmodium falciparum, six P. knowlesi and one Plasmodium vivax . Of the six P. knowlesi deaths, five were attributable to knowlesi malaria and one was attributable to P. knowlesi-associated enterobacter sepsis. Patients with directly attributable P. knowlesi deaths (N = 5) were older than those with P. falciparum . Complications in fatal P. knowlesi included respiratory distress , hypotension , and renal failure . All patients with P. knowlesi were reported as P. malariae by microscopy. Only two of five patients with severe knowlesi malaria on presentation received immediate parenteral anti-malarial treatment. The patient with P. vivax-associated severe illness did not receive parenteral treatment. In contrast six of seven patients with severe falciparum malaria received immediate parenteral treatment.Fourteen malaria deaths were reported, comprising seven Plasmodium knowlesi was responsible, either directly or through gram-negative bacteraemia, for almost half of malaria deaths in Sabah. Patients with severe non-falciparum malaria were less likely to receive immediate parenteral therapy. This highlights the need in Sabah for microscopically diagnosed P. malariae to be reported as P. knowlesi to improve recognition and management of this potentially fatal species. Clinicians need to be better informed of the potential for severe and fatal malaria from non-falciparum species, and the need to treat all severe malaria with immediate intravenous artesunate. Plasmodium knowlesi is commonly misdiagnosed as Plasmodium malariae by microscopy due to its near-identical appearance. However, in contrast to the relatively benign clinical course of P. malariae, P. knowlesi is now recognized as a common cause of severe and fatal human malaria in Malaysian Borneo [P. knowlesi mono-infection accounted for 64% of malaria admissions in Kapit, Sarawak [The simian parasite n Borneo . Cases hn Borneo  and nearn Borneo -10. In B Sarawak , and 78% Sarawak . Severe  Sarawak , Sabah [ Sarawak ,14, and  Sarawak , includi Sarawak ,13,14. IP. knowlesi may be associated with rapid increases in parasitaemia and consequent complications, and hence prompt diagnosis and initiation of effective treatment is essential. The optimal treatment however has not been determined. While chloroquine was shown to be effective for uncomplicated knowlesi malaria in Kapit [The 24-hour replication cycle of in Kapit , the retin Kapit . Among pin Kapit .Plasmodium species causing fatal malaria in Sabah, and to identify any notable differences in demographics, clinical features and management of fatal malaria caused by the different species.In Sabah, intravenous artesunate has been the recommended treatment for severe malaria from any species since December 2008. In addition, referral to a general hospital is advised for patients with symptoms or signs suggestive of severe disease. Despite these measures, 14 deaths from malaria were reported in Sabah during 2010-2011. In this study the case notes of all these patients were reviewed to determine the 2 and a population of 3.2 million [The north-eastern Malaysian state of Sabah has an area of 73,600 km million . Situate million . Sabah’sAll deaths due to malaria in Sabah must be reported to the Sabah Ministry of Health where they are reviewed. Details of reported malaria deaths during 2010-2011 were obtained from the Sabah Department of Health. Approval to review the case notes of fatal malaria cases was obtained from the Medical Research sub-Committee of the Malaysian Ministry of Health and the Health Research Ethics Committee of Menzies School of Health Research. Case notes were retrieved from district hospitals and reviewed for clinical details, laboratory results and cause of death.Blood slides for malaria parasites were reported according to a scale of 1+ to 4+ . PCR was performed by the Sabah State Reference Laboratory by methods previously published . The diaP. knowlesi mono-infection, one P. knowlesi-associated fatality in which gram-negative septic shock was thought to be the primary cause of death, one death associated with P. vivax and seven from P. falciparum.Fourteen deaths were reported during 2010-2011, including five due to Plasmodium falciparum/P. malariae “3+”, and renal failure was present. Chest radiograph showed no infiltrates, and arterial blood gas was not performed. He was commenced on intravenous fluids and quinine in addition to oral primaquine, chloroquine and doxycycline. His oxygen saturation and blood pressure continued to deteriorate and he suffered a cardiac arrest 40 minutes after presentation. PCR confirmed P. knowlesi mono-infection.A 71-year-old man with a history of hypertension and chronic obstructive pulmonary disease presented to a district hospital with a seven-day history of fever and dyspnoea, and reduced conscious state just prior to presentation. His Glasgow Coma Score (GCS) was recorded on arrival by a paramedic as 3/15 and shortly afterwards as 10/15; his blood pressure was 75/49 mmHg and his oxygen saturation was 75% on room air. Chest auscultation noted prolonged expiratory phase but no crackles or wheeze. Blood film was reported as P. malariae “3+” and oral chloroquine was started. The following day the patient was noted to be tachypnoeic and hypoxic (oxygen saturation 85% on room air), and chest radiograph showed diffuse infiltrates. Emergency intubation was performed however cardiac arrest occurred during the procedure. Cause of death was reported as severe malaria with acute respiratory distress syndrome.A 65-year-old man with a history of type 2 diabetes and hypertension presented to a district hospital with a two-day history of fever, lethargy, myalgia and retro-orbital pain. On examination he was alert and orientated but hypoxic with an oxygen saturation of 92% on room air. Initial blood film was reported as negative for malaria parasites, and the patient was given a provisional diagnosis of dengue fever and commenced on intravenous fluids. On day 2 a repeat blood film was reported as P. malariae “4+”, and he had renal failure with a creatinine of 578 μmol/L. Chest radiograph revealed patchy consolidation in both lung fields. He was commenced on intravenous fluids, oxygen supplementation, inotropic support, intravenous antibiotics and intravenous artesunate, and transferred to a general hospital, where he was intubated and ventilated and commenced on haemodialysis. He died the following day from acute respiratory distress syndrome. Blood cultures were negative.A 51-year-old man with no known medical history presented to a clinic with a four-day history of fever, rigours, myalgia and arthralgia. On examination he was drowsy, jaundiced, hypotensive (blood pressure 70/40 mmHg), tachycardic (heart rate 110 beats/minute) and hypoxic (oxygen saturation 90% on room air). Hepatomegaly was noted on abdominal examination. Blood film was reported as Plasmodium vivax “4+”. The patient was commenced on intravenous fluids, antibiotics and oral chloroquine, and transferred to a general hospital, where he was intubated and ventilated, and commenced on inotropic support. Chest radiograph showed diffuse infiltrates. The patient had renal failure (creatinine 330 μmol/L) and severe thrombocytopenia (3 x 103 platelets/μL) although no bleeding complications were noted. Haemodialysis was commenced and platelet transfusion given. On day 3 a repeat blood film was reported as P. malariae “4+”. Intravenous artesunate was commenced, however the patient remained on maximum inotropic and ventilator support, and further haemodialysis was temporarily unavailable. He died nine hours later with multiple organ failure. Cause of death was reported as severe malaria. Blood cultures and dengue serology were negative, and PCR performed on a blood sample taken on day 3 confirmed P. knowlesi monoinfection.A 50-year-old man with no known medical history presented to a district hospital with a seven-day history of fever and rigours and a three-day history of cough. On examination he was alert and orientated but hypotensive (blood pressure 83/51 mmHg) and hypoxic (oxygen saturation 70% on 10 L oxygen via high flow mask), and wheeze was heard on chest auscultation. Blood film was reported as P. malariae “4+”, and renal failure was present (creatinine 283 μmol/L). Bronchodilators, intravenous antibiotics, oral chloroquine and primaquine were commenced in addition to inotropic support and transfer to a tertiary hospital. Chest radiograph on arrival revealed bilateral lower zone infiltrates, and the patient was intubated and ventilated however developed refractory hypotension and died the following morning. Cause of death was stated as septic shock, although blood cultures were not performed.A 49-year-old woman with no known medical illness presented to a district hospital with a four-day history of fever, cough and dyspnoea. On examination she was tachypnoeic and hypoxic (oxygen saturation on room air 88%) and wheeze was heard on chest auscultation. Blood film was reported as P. malariae “1+” and she was treated with oral sulphadoxine/pyrimethamine (SP) and primaquine. On day 3, she was aparasitaemic, but had become tachypnoeic (respiratory rate 44 breaths/minute), hypoxic (oxygen saturation 85% on room air) and hypotensive, and widespread wheeze was heard on chest auscultation. Chest radiograph showed no infiltrates and arterial blood gas revealed metabolic acidosis . She was commenced on bronchodilators, intravenous ceftriaxone and inotropic support and transferred to a tertiary hospital where she was intubated and ventilated and given intravenous artesunate. Further investigation results at the tertiary hospital included haemoglobin 8.0 g/dL, platelets 65 x 103/μL, white cell count 6.6 x 103/mL, creatinine 149 μmol/L and aspartate aminotransferase 795 U/L. Following admission the patient’s blood pressure deteriorated further and she developed a tachyarrthymia. Cardioversion was unsuccessful and the patient died from cardiac arrest four hours after arrival. Cause of death was reported as severe malaria, however on review, blood cultures taken on admission to the district hospital were noted to be positive for Enterobacter cloacae (reported as sensitive to ceftriaxone), untreated until progression to septic shock. Repeat cultures taken after antibiotics at the referral hospital were negative.A 36-year-old woman with no known medical illness presented to a district hospital with a seven-day history of fever, cough and myalgia. Examination was unremarkable and no features of severe malaria were evident. Blood film was reported as 3/μL) and acute kidney injury . Blood film was reported as P. malariae “4+”, and oral chloroquine was commenced. Her condition deteriorated with increasing oxygen requirements and on day 3 her blood pressure became unrecordable. The cause of death was reported as dissecting aortic aneurysm. PCR identified P. vivax mono-infection.An 85 year-old-woman presented to a district hospital with fever, rigours and abdominal pain. On examination she was pale, tachypnoeic and hypoxic with oxygen saturation of 88% on room air. Her blood pressure was 137/88 mmHg, and chest auscultation was clear. No arterial blood was available. On abdominal examination a tender pulsating mass was noted, and bedside ultrasound confirmed a 7.6 cm x 5.6 cm abdominal mass with minimal free fluid. No computed tomography scan was done. A provisional diagnosis of aortic dissection was made, and after discussion with family, conservative management was planned. Blood investigations revealed anaemia (haemoglobin 8.8 g/dL), thrombocytopenia  and four adults (aged 22–60 years). All the children and two adults were Filipino. All met the WHO criteria for severe malaria  on preseP. knowlesi as P. malariae. It also highlights the lack of recognition of non-falciparum Plasmodium species as potential causes of severe and fatal malaria, and the fatal consequences of initial oral therapy for severe malaria of any species, particularly P. knowlesi.This case series highlights the misdiagnosis of severe knowlesi malaria due to continued reporting of P. knowlesi were older than those with fatal P. falciparum . The older age group affected, and the complications experienced by the fatal knowlesi malaria patients, are consistent with those already reported [In this series patients with deaths directly attributable to reported ,14. In preported ,14, and reported ,14, and P. knowlesi. Pre-antibiotic blood cultures were taken in three of these patients and were all negative. Relatively low rates of clinically significant bacteraemia have been reported previously in severe knowlesi malaria [P. knowlesi. Nevertheless, one patient in this series who presented with uncomplicated knowlesi malaria had Enterobacter cloacae bacteraemia on admission, with bacterial septic shock the likely cause of death. This species was also identified in the only other report of clinically significant bacteraemia in a patient with severe knowlesi malaria [Salmonella due to malaria-induced haemolysis and neutrophil dysfunction [P. falciparum in gut mucosa, and parasite accumulation in multiple organs was demonstrated in the single P. knowlesi autopsy report. Further studies are required to investigate mechanisms of bacteraemia in P. knowlesi infection.Hypotension is also a common complication of severe knowlesi malaria, and occurred in four of five (80%) patients with deaths directly attributable to  malaria , suggest malaria . Gram-ne malaria -25. The function , impairefunction -29, and function . BacteriComa has not been reported to occur in knowlesi malaria, despite the only autopsy report demonstrating cerebral accumulation of parasitized red blood cells . One patP. knowlesi has a 24-hour replication cycle, which can lead to rapid increases in parasitaemia. Diagnosis of this malaria species is therefore critical in order that its potential to cause severe disease is recognized, appropriate treatment instituted without delay, and further complications and fatalities avoided. In this series, no patient was correctly diagnosed with P. knowlesi. Rather, all received a diagnosis of P. malariae, despite a very low incidence of this species in Sabah, with P. malariae detected by nested PCR in only 2 of 318 (0.6%) microscopy-diagnosed P. malariae cases referred to the Sabah State Public Health Laboratory in 2009 [P. knowlesi is indistinguishable from P. malariae. While there may be very subtle morphological differences between the late stages of P. knowlesi and P. malariae on thin film microscopy, these are not consistent and cannot be relied upon in clinical practice [P. malariae causes a relatively benign acute illness, with low parasitaemia and only very rare reports of acutely severe malaria [P. malariae to be reported as P. knowlesi in Sabah, in order to direct clinicians in the recognition and management of this potentially fatal species.In contrast to other human malarias, practice . P. mala malaria ,34. CurrPlasmodium knowlesi is also sometimes misdiagnosed as P. vivax  and P. vivax can also cause severe and fatal disease [P. vivax should also be treated with intravenous artesunate if signs of severity are present, particularly as oral chloroquine may be poorly absorbed in acutely unwell patients and chloroquine-resistant P. vivax is increasingly prevalent in Southeast Asia [P. knowlesi, has been Sabah state policy since 2008 and has been adopted in the latest WHO severe malaria management guidelines [P. falciparum vs non-falciparum; Yates corrected Chi2). These findings suggest a lack of recognition of the potential of the non-falciparum malarias to cause severe disease and the need for treatment with intravenous artesunate for all severe malaria patients.Moreover, this case series illustrates the importance of a unified treatment policy for all patients with severe malaria, regardless of species . Althoug disease ,37. For ast Asia . This unidelines . DespiteThis study had several limitations, the major one being its retrospective design resulting in unavoidably incomplete laboratory and clinical data. Blood culture results were not available for two patients, and for these patients bacterial sepsis cannot be excluded as contributing to poor outcome. Lack of accurate parasite density counts also made assessment of parasite burden difficult. Post-mortem examination was not performed for any patient, preventing detailed description of the pathogenic mechanisms of severe malaria and death.Plasmodium knowlesi is a common cause of malaria in Sabah, and in this series was responsible, either directly or through bacteraemia, for almost half of malaria deaths throughout the state. These results highlight the importance of accurate reporting of P. knowlesi to improve recognition and management of this species. Clinicians need to be better informed of the potential for severe and fatal malaria from non-falciparum species, especially P. knowlesi, and the need to treat all severe malaria with immediate intravenous artesunate regardless of species.The authors declare that they have no competing interests.GR performed the case note review with assistance from TW and BB. TW and JM facilitated the case review. GR, BB, NA and TY wrote the paper. All authors read and approved the final manuscript.


However, their use in South America has been widely questioned following a recent publication that pinpoints the high prevalence of Peruvian field isolates lacking the gene encoding this protein. In the remote rural health centers of French Guiana, RDTs are the main diagnosis tools. Therefore, a study of PfHRP2 RDT performances and pfhrp2 genotyping was conducted to determine whether a replacement of the current pLDH-based kit could be considered.Care for malaria patients in endemic areas has been improved through the increasing use of Rapid Diagnostic Tests (RDTs). Most RDTs target the histidine-rich protein-2 antigen , and 86.0% (95% CI: 78.9–91.5) for the detection of P. vivax. No isolates (95% CI: 0–4.5) lacking either exon of the 2pfhrp gene were identified among the 221 P. falciparum isolates analyzed, but 7.4% (95% CI: 2.8–15.4) lacked the exon 2 part of the 3pfhrp gene.Between January 2010 and August 2011, 960 suspected cases of malaria were analyzed using microscopy and RDTs. The sensitivity of the SD Malaria Ag test P.f/Pan® for detection of 2pfhrp gene are absent in this western part of South America. Despite its sensibility to detect P. vivax, the SD Malaria Ag test P.f/Pan® kit is a satisfying alternative to microscopy in remote health centers, where it is difficult to provide highly skilled microscopists and to maintain the necessary equipment.Field isolates lacking either exon of the  In the late 1990’s, the introduction of Rapid Diagnosis Tests (RDT) for malaria provided a rapid, accurate and accessible diagnostic tool that reduced clinical diagnosis in endemic areas where skills and materials are lacking. As malaria symptoms are non-specific, this lowered presumptive treatment and therefore reduced drug pressure on the parasite population. Numerous commercial kits exist. WHO therefore evaluated the performances of a wide panel of commercial kits, using a standardized procedure in order to compare performances between them. The results of these evaluations have been published and included in general guidance for health workers P. falciparum and P. vivax represented 31% and 68.5% of the 1,205 cases, respectively P. malariae cases are occasional. Transmission occurs along thousands of miles of rivers with a low number of focal points in the coastal area where the majority (90%) of the population lives. This transmission is closely linked with the mostly illegal gold mining activities French Guiana is an outermost region of France located in Amazonia between North Brazil and Suriname. The health system in this region consists of a network of medical structures that allow patients to be treated close to their homes. The Hospital of Cayenne, the main town of French Guiana, administers these rural health-posts, which are spread over the entire territory. In the smallest of these healthcare facilities, the health provider team is composed of just one nurse. In this Amazonian area, malaria is endemic. In 2011, P. falciparum, PfHRP2), Lactate Dehydrogenase (pLDH) or aldolase. P. falciparum secretes three histidine-rich proteins that share repeats of hexapeptides representing the epitopes, and they are recognized by monoclonal antibodies (Mab) raised against PfHRP2 protein. Cross-reactions may therefore occur between the PfHRP2 specific Mab and epitopes of the PfHRP3 protein. These hexapeptides are encoded by the exon 2 part of the gene, which is very polymorphic among isolates P. falciparum; pLDH-pv for P. vivax) P. falciparum, and the recent commercialization of several new kits, the authorities are currently considering switching to the pLDH-pan+PfHRP2 kit. HRP2-based RDTs are indeed more suitable for the diagnosis of P. falciparum because of their higher sensitivity, better stability across a wider temperature range and lower cost RDTs for malaria are specific for one of the three following antigens: Histidine Rich Protein 2 , which was wrongly diagnosed by HRP2 RDTs as “species different from P. falciparum” and treated by chloroquine. Owing to the prevalence of chloroquino-resistant parasites (25%) in this region, this could have had serious consequences for the patient pfhrp2 gene and pfhrp3 exon 2) in parasites from French Guiana and its consequences on performances of an HRP2-based RDT, the SD Malaria Ag P.f/Pan® kit.Recently, up to 41% of parasites in Peru were reported to lack the pfhrp2 gene deletion, we first performed a retrospective study on a total of 140 P. falciparum isolates collected throughout French Guiana in 2009  for Malaria, the parasitology laboratory of the Institut Pasteur de la Guyane is responsible for collecting the majority of malarial samples drawn in French Guiana in order to follow the resistance level of the parasites to antimalarial drugs. In an attempt to evaluate the prevalence of the  in 2009 . Isolate2 based RDT, SD Malaria Ag P.f/Pan® (Standard Diagnostics Inc.). For this purpose, we included febrile patients of all ages who consulted for suspected malaria during a prospective study. For a proper assessment of performances , the number of cases required in each of the three groups  was evaluated at 73, and recruitment continued until at least the required number was reached for each group.In a second step, we evaluated the performances of the PfHRPVenous blood for malaria diagnosis was taken from 4 ml samples collected in EDTA tubes.Reference method. Microscopic examination by an expert microscopist was considered the reference method for malaria diagnosis. Thick and thin blood films were prepared within one hour of blood collection. Thick blood films were stained with Giemsa diluted at 10%, whilst thin blood films were stained using a rapid method . Two hundred fields of the thin blood film were examined before classifying the thin smear negative, then 1000 counted white blood cells (WBCs) from the thick smear were observed before classifying the sample as negative. The parasite density estimation was based on an assumed 6,000 WBC/µl of blood.The microscopic examination results were transmitted to the appropriate physicians within an hour of the blood being drawn. In the case of positive results, patients were treated following the regional guidelines for malaria Rapid diagnostic tests. The SD Malaria Ag P.f/Pan® (Standard Diagnostics Inc.) detects the presence of pan-pLDH and PfHRP2 antigens. This test was performed according to the manufacturer’s instructions, and the microscopic examination was carried out simultaneously by a second technician. Interpretation of the test results was carried out independently of the microscopic examination and within the time recommended by the manufacturer. Test results were considered invalid if no control band was seen. The tests were guaranteed to have been correctly stored (temperature 24–28°C) and were used within their recommended shelf life.P. falciparum and P. vivax, respectively (data not shown).In the case of diagnosis discrepancies between RDT and microscopy, a PCR method was used to determine the species present in the sample. DNA was extracted from 200 µl of venous blood collected from EDTA tubes using the QIAmp® DNA mini kit (Qiagen). All the manufacturer’s instructions were followed with the exception of the elution step, which was carried out with 100 µl of elution buffer instead of 200 µl. A nested-polymerase chain reaction (PCR) was performed for samples with non-concordant results. Species-specific nested PCR tests were carried out for each of the four main malaria species, as previously described P. malariae cases were removed from the study. Mixed P. falciparum/P. vivax infections were analyzed in the P. falciparum group, as the SD Malaria Ag P.f/Pan® only allows the distinction between P. falciparum and other species. The performances of the SD Malaria Ag P.f/Pan® kit were expressed by calculating the sensitivity and the specificity for P. vivax and P. falciparum separately, taking microscopy results as the “gold standard”.P. falciparum or P. vivax samples showing concordant microscopy and RDT results divided by the total number of P. falciparum or P. vivax results gained through microscopy, respectively.Sensitivity is the ability of the test to detect parasites among samples seen to contain parasites during microscopic examination. The sensitivity value was the number of P. falciparum or P. vivax  divided by the total number of negative P. falciparum or P. vivax microscopy results, respectively.Specificity is the ability of the test to correctly classify negative samples among negative microscopy results. The specificity value was the number of samples lacking Data were analyzed using the Epi-info 3.5.1 software . Results were accompanied by 95% confidence intervals (95% CI) calculated using the Clopper Pearson exact method pfhrp2 gene and the exon 2 of the pfhrp3 gene from 2 µl of DNA using the primers and conditions described in pfhpr2 gene from 10 bp before the start codon to 305 bp after; the A118/A119 primer pair from 225 bp after the start codon to 274 bp after the stop codon of the pfhrp2 coding sequence and A266/A261 amplified the exon 2 part of the pfhrp3 gene from 226 bp after the start codon to 20 bp after the stop codon. PCR products were analyzed by electrophoresis in 1.5% agarose gel. Sensitivities of these PCRs were evaluated at 25 and 50 parasites/µl of whole blood, respectively (data not shown).We amplified either exon of the pfhrp2 gene was sequenced for a panel of 30 P. falciparum isolates identified during the prospective study. These were randomly selected with the exception of M618, the isolate of particular interest. The PCR products obtained after the amplifications, with the primer pairs A118/A264 and A262/A263 were purified using spin columns (QIAGEN) then sequenced using standard dye-terminator sequencing reactions (Applera). The sequences reported in this article have been deposited in the GenBank database (accession number KC558574 to KC558602). Nucleotide sequences were translated to amino acids and each repeat was identified using a previously-described numeric code system The Analyzed samples were all obtained by blood collections required by the standard medical care for any patient presenting fever on hospital admission in French Guiana. According to the French legislation , biobanking and secondary use for scientific purpose of human clinical remaining samples are possible as long as the corresponding patients are informed and has not given any objection to them. In the present research, this requirement is fulfilled: information is given to every patient through the Hospital brochure entitled “Information for patients”, and no immediate or delayed patient opposition was reported by the hospital clinicians to the Malaria NRC.Moreover, in application of French legislation , samples received at the Malaria NRC had been registered for research purpose in the NRC biobank declared to both the French Ministry for Research and a French Ethics Committee which both approved and registered this thematic biobank . No institutional review board approval is required according to the French legislation.pfhrp2 gene of 140 P. falciparum isolates collected from different parts of the country in 2009  isolates lacked the pfhrp3 exon 2.We amplified either exon of the  in 2009 . The M61P. malariae cases were excluded and the 7 P. falciparum/P. vivax mixed-infections detected by microscopy were analyzed as P. falciparum infection. Microscopic examination revealed the majority of the samples  to be negative for malaria, whilst 93 (9.7%) and 129 (13.4%) isolates were positive for P. falciparum and P. vivax, respectively  were observed. The SD Malaria Ag P.f/Pan® kit exhibited a sensitivity of 96.8% (95% CI: 90.9–99.3) to detect P. falciparum and 86.0% (95% CI: 78.9–91.5) to detect P. vivax. The associated specificities were 98.8% (95% CI: 97.9–99.5) and 99.6% (95% CI: 99.0–99.9) for the diagnosis of P. falciparum and P. vivax, respectively.The prospective study to evaluate the performances of the SD Malaria Ag P.f/Pan® kit was conducted from July 2010 to July 2011. During this period, 960 EDTA blood samples were collected at Cayenne Hospital from patients with suspected malaria. The male/female ratio was 1.2∶1 with a median age of 25.8 years [range 1–92 years]. The place of contamination was unknown for most of the patients. According to the data analysis plan, three ectively . The medP. vivax isolates. These false negative results were essentially linked to isolates with a parasite density of less than 0.01% (n = 12/14).Twenty-eight discrepant results were found between the microscopic examination and RDT results . They wepfhrp2 genotype of the parasites, one isolate of interest was identified, namely N500. PCR validation and a second microscopic examination determined that it was a mixed infection (Pf/Pv) missed by both microscopy and the RDT kit. In this isolate, the pfhrp3 exon 2 was deleted, whilst either exon of the pfhrp2 gene was present.Regarding the discrepancies potentially linked to the pfhrp2 gene, the 93 confirmed P. falciparum samples of the prospective study were included in a systematic presence/absence of the entire pfhrp2 gene and pfhrp3 exon 2 genotyping processes. Within these 93 isolates, ten DNA were not available for analysis, and two isolates were excluded after PCR confirmed them to be either negative or to contain P. vivax  of cases, a deletion of the exon 2 part of the pfhrp3 gene was observed.As the isolates included in the retrospective study did not contain any parasites lacking either exon of the P. vivax . None , and the M618 isolate was part of this group. No polymorphism was observed in the exon 1 part of the pfhrp2 gene while variability in the microsatellite included in the intron 1 was observed in two samples (16 and 18 repeats versus 15 for the others). Furthermore, no frame shift in the pfhrp2 sequence of these samples was observed.For a better understanding of the PfHRP2 polymorphism displayed by the isolates circulating in French Guiana, 30 samples were genotyped. Twenty-nine larities . The gropfhrp2 gene and only 4.5% lacked the pfhrp3 gene.The present study is the first report carried out in the eastern part of the Amazonian region to simultaneously assess: i) the genotype of the histidine-rich protein gene family, and ii) the associated performances of an HRP2-based RDT kit. It confirms that these types of RDT are suitable for malaria diagnosis in French Guiana, as none of the parasites in this part of the world lack the whole P. falciparum diagnosis should provide a sensitivity of over 95% P. falciparum and P. vivax, respectively. Comparison with previous studies is difficult, as test performances may vary due to i) different geographical variations of parasite populations, ii) levels of disease prevalence and iii) the study design P. falciparum correlates with previous results obtained with other PfHRP2 based kits in the region P. vivax. This value is mainly explained by the false negative results obtained for isolates with parasitemia values of under 0.01%. P. vivax cases being generally more benign than P. falciparum, the SD Malaria Ag P.f/Pan® kit could therefore be considered suitable for malaria diagnosis in French Guiana.In order to be reliable, a method for pfhrp2 gene deletion  and 4.5% deletion of the pfhrp3 exon 2 . This sampling represented 14.8% (221/1495) of the total numbers of P. falciparum malaria cases identified in French Guiana on the same period. Even if the majority of the isolates were collected in Cayenne, a non-endemic town, local knowledge on human population movements suggests that malaria samples collected in this town are from different parts of French Guiana. Therefore, these results could be extended to the general parasite population circulating in the region between 2009 and 2011. This absence of pfhrp2 deletion contrasts with a previous study in the Amazonian region of Peru pfhrp2 gene and more than 70% for the pfhrp3 gene. These results, associated with the very high prevalence of P. vivax in South America, led WHO to recommend the pLDH-based RDTs in this part of the world. However, results from other parts of the continent are scarce If we consider the entire set of analyzed samples, these performances were associated with an absence of P. falciparum in French Guiana and is probably linked to the low transmission level of this species within this endemic area A study was carried out on the genetic variability of this gene, including exon 2 which encodes the epitopes detected by the RDTs. No frame shift and a limited genetic diversity were observed among the 29 analyzed samples. Haplotype 1 was predominant in 82.3% of the isolates. This low level of diversity correlates with the general diversity of P. falciparum isolate was of particular interest as two different PfHRP2-based  kits failed to diagnose it, yet it did not lack the whole pfhrp2 gene, neither the exon 2 part of the pfhrp3 gene. The possibility of a prozone effect due to an excess of either antigen or antibodies was excluded after testing a 1/10 and 1/100 dilution of the sample (data not shown) pfhrp2 sequence did not exhibit a frame shift, or particular mutations. Its haplotype was part of the 1 d group composed of two isolates. The second isolate from this group, with a parasitaemia of 0.66%, was detectable by the SD Malaria Ag P.f/Pan®. With a parasitaemia of 0.4%, this failure of detection of the M618 could not therefore be explained by inadequate kit sensitivity. Then, we calculated the product of the number of type 2 × type 7 repeats as previously described to classify the isolates as sensitive or not to detection by a PfHRP2 RDT in vitro propagation or available RNA extracts.In our study, the M618 pfhrp2 and pfhrp3 deletions among isolates from other countries located in this region would permit us to verify this assumption. In the absence of such studies and as parasites lacking the pfhrp2 gene exist on the continent; a regular control of their prevalence is required.This study provides important information for laboratories and health policy makers, as it demonstrates that PfHRP2-based RDTs are suitable for malaria diagnosis in French Guiana. At a regional level, human population migration and current knowledge of parasite diversity  lead us to believe that this situation should be the same throughout the Guiana shield