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Additional file 1: Material S1. Anti-malarial treatment details for IBSM individuals. Table S1. Antimalarial treatment given for the 15 IBSM individuals from four different studies, as identified with the clinical trial name and clinical trial ID. The antimalarial and dose given at day of treatment on Day 7 is detailed for each subject. Material S2. Methods to fit PfHRP2 model to the 6 individuals from the Namibia longitudinal cohort study. Table S2. Model performance of the Base model and red the Final model for the 15 IBSM individuals. Performance measures were the number (%) of PfHRP2 observations within the range of the predicted minimum and maximum PfHRP2 concentrations, the residual sum of squares (RSS) and the residual mean sum of square (RMSE). Table S3. Model performance of the updated model with PfHRP2 half-life estimate of 1.67 days applied to 6 individuals aged between 23 and 27 years from the study in Namibia in participants with Plasmodium falciparum mono-infection. Performance measures were the number (%) of PfHRP2 observations within the range of the predicted minimum and maximum PfHRP2 concentrations, the residual sum of squares (RSS), residual mean sum of square (RMSE), the predicted and observed days above 800 or 80 pg/mL. Figure S1. Fits of the Base Model and the Final Model for the 15 IBSM individuals. The observed parasitemia over the course of infection is represented by black solid line, observed PfHRP2 concentration is represented by circles (pre-treatment in solid circles and post-treatment in open circles), and the predicted minimum and maximum PfHRP2 concentration from the model is shown as blue and red dashed lines, respectively. Figure S2. Simulated parasitemia growth and clearance over the course of the infection for each of the 6 individuals from the Namibia longitudinal cohort study is represented by the black line. The observed parasitemia (parasites/mL) during the study are represented by the blue triangles and the replicating parasites used as input into the PfHRP2 model are represented by open circles. Figure S3. Fits of the final model with elimination half-life of 1.67 days to the 6 individuals from the Namibia longitudinal cohort study, with observed PfHRP2 concentration represented by closed circles, and minimum and maximum predicted PfHRP2 concentration represented by the blue and red dashed lines, respectively. The dashed grey horizontal line represents the threshold of 800 pg/mL and the solid grey horizontal line represents the threshold of 80 pg/mL which correspond to the positivity threshold of an RDT and usRDT respectively.

Additional file 1. Estimating population-level prevalence by HS-RDT and PCR in Owalla et al.

Additional file 1: Table S1. Comparison of two multiplex platforms for the quantitative assessment of malaria antigens. Table S2. Characteristics of the Q-Plex and xMAP assays. Table S3. Linear relation between the WHO international standard P. falciparum and P. vivax antigens and assay concentration results in whole blood or DBS. Regression analysis showed the fit of the data with R2 values that met the acceptance criteria (R2 ≥ 0.85). One operator conducted three experiments over a week. Figure S1. HRP2 concentration in (A) whole blood and (B) DBS plotted against the parasitemia. Three subsets of dilution-series samples composed of different P. falciparum laboratory strains (W2, Dd2, and 3BD5) were tested by using the Q-Plex and xMAP. Measured concentration was plotted on the y-axis against parasitemia (par/mL) on the x-axis. Red and blue symbols and regression lines represent samples spiked with different strains and test types. Figure S2. Correlation analysis. Correlation plots of antigen concentration corrected against the WHO international standard P. falciparum and P. vivax antigens, P. falciparum culture, and clinical samples in (A) whole blood and (B) DBS. The Pearson correlation r value and the equation of the regression line obtained from analysis using log transformed data are shown. Outlier sample sets were color-coded.

Additional file 4. Correlation between parasite density and HRP2 concentration, and positivity by HS-RDT and co-RDT.

Additional file 2. Positive and negative predictive values for the HS-RDT and co-RDT.

Additional file 1: Figure S1. Scatterplot showing correlation between parasite count by microscopy and HRP2 concentration quantified by Q-Plex ELISA (R2 = 0.29) for all samples P. falciparum positive by microscopy (> 0 parasites/µL). Abbreviations: ELISA, enzyme-linked immunosorbent assay; HRP2, histidine rich protein 2; pLDH, Plasmodium lactate dehydrogenase. Figure S2. Scatterplot showing correlation between parasite count by microscopy and pLDH concentration quantified by Q-Plex ELISA (R2 = 0.61) for all samples P. falciparum positive by microscopy (> 0 parasites/µL). Table S1. Antigen ratios by direct skin feed (DSF) infectivity status. Geometric means for the two antigens of interest, HRP2 and pLDH, along with the geometric mean HRP2:pLDH ratio for study participants that were DSF-positive (n = 11) or DSF-negative (n = 603), grouped by microscopy result. Individuals positive by DSF had a significantly lower HRP2:pLDH ratio (p = 0.02) on average in reference to DSF and microscopy negative individuals. Abbreviations: geo, geometric; HRP2, histidine rich protein 2; pLDH, Plasmodium lactate dehydrogenase; SD, standard deviation.

Background:\ud Plasmodium malariae is considered a ‘minor’ malaria parasite, although its global disease burden is underappreciated. The aim of this study was to develop an induced blood stage malaria (IBSM) model of P. malariae to study parasite biology, diagnostics, and treatment.\ud \ud Methods:\ud This clinical trial involved two healthy subjects who were intravenously inoculated with cryopreserved P. malariae-infected erythrocytes. Subjects were treated with artemether-lumefantrine following development of clinical symptoms. Prior to antimalarial therapy, mosquito feeding assays were performed to investigate transmission, and blood samples were collected for rapid diagnostic testing and parasite transcription profiling. Serial blood samples were collected for biomarker analysis.\ud \ud Results:\ud Both subjects experienced symptoms and signs typical of early malaria. Parasitaemia was detected 7 days post-inoculation and increased until antimalarial treatment was initiated 25 and 21 days post-inoculation for Subject 1 and 2 respectively (peak parasitaemia levels were 174,182 and 50,291 parasites/mL respectively). The parasite clearance half-life following artemether-lumefantrine treatment was 6.7 hours. Mosquito transmission was observed for one subject, while in vivo parasite transcription and biomarkers were successfully profiled.\ud \ud Conclusions:\ud An IBSM model of P. malariae has been successfully developed and may be used to study the biology, diagnostics, and treatment of this neglected malaria species.

G6PD deficiency is a common X-linked genetic trait, with an associated enzyme deficiency phenotype, whereby males are either G6PD deficient or normal, but females exhibit a broader range of G6PD-deficiency, ranging from severe deficiency to normal. Heterozygous females typically have intermediate G6PD enzyme activity levels. In the malaria field, G6PD deficiency has significant implications for the safe and appropriate treatment for P.vivax malaria, as individuals with this deficiency are at greater risk of serious adverse events following treatment with the only curative class of anti-malarials, 8-aminoquinolines, such as primaquine. To date, the gold standard quantitative diagnostic tests for measuring G6PD deficiency are complex and require sophisticated laboratories. Qualitative tests, which are most commonly used, do not discriminate intermediate G6PD activity ranges. This has resulted in poor understanding of the epidemiology of G6PD activity in heterozygous females and its corresponding treatment ramifications. With the advent of simple-to-use quantitative tests for G6PD deficiency, and a momentum to eliminate malaria, there is an opportunity to address this knowledge gap. While this will require additional resources for clinical studies, adequate operational research, and appropriate pharmacovigilance, the health benefits from this investment go beyond the immediate intervention for which the G6PD status is first diagnosed.

Background Medicines that exert oxidative pressure on red blood cells (RBC) can cause severe hemolysis in patients with glucose-6-phosphate dehydrogenase (G6PD) deficiency. Due to X-chromosome inactivation, females heterozygous for G6PD with one allele encoding a G6PD deficient protein and the other a normal protein produce two RBC populations each expressing exclusively one allele. The G6PD mosaic is not captured with routine G6PD tests. Methods An open-source software tool for G6PD cytofluorometric data interpretation is described. The tool interprets data in terms of % bright RBC, or cells with normal G6PD activity in specimens collected from two geographically and ethnically distinct populations, an African-American cohort (US), and a Karen and Burman ethnic cohort (Thailand) comprising 242 specimens including 89 heterozygous females. Results The tool allowed comparison of data across two laboratories and both populations. Hemizygous normal or deficient males and homozygous normal or deficient females cluster at narrow % bright cells with mean values of 96%, or 6% (males) and 97%, or 2% (females) respectively. Heterozygous females, show a distribution of 10-85% bright cells, and a mean of 50%. The distributions are associated to severity of the G6PD mutation. Conclusions Consistent cytoflourometric G6PD analysis facilitates inter-laboratory comparison of cellular G6PD profiles and contributes to understanding primaquine associated hemolytic risk.

Glucose-6-phosphate dehydrogenase (G6PD) activity is essential for redox equilibrium of red blood cells (RBCs) and, when compromised, the RBCs are more susceptible to haemolysis. 8-aminoquinolines (primaquine and tafenoquine) are used for the radical curative treatment of Plasmodium vivax malaria and can cause haemolysis in G6PD deficient subjects. Haemolytic risk is dependent on treatment dose and patient G6PD status but ultimately it correlates with the number of G6PD deficient RBCs. The G6PD spectrophotometric assay reliably identifies deficient subjects but is less reliable in heterozygous females, especially when other blood conditions are present. In this work we analysed samples with a range of G6PD phenotypes and haematologic conditions from 243 healthy volunteers of Asian or African-American heritage using both the spectrophotomeric assay and the G6PD flow-cytometric assay. Overall 18.5% of subjects (29.3% of Asian females) presented with anaemia, associated with decreased RBCs volume (MCV) and reticulocytosis; the flow-cytometric assay showed good correlation with the spectrophotometric assay (Pearson’s r 0.918–0.957) and was less influenced by haemoglobin concentration, number of RBCs and number of reticulocytes. This resulted in more precise quantification of the number of G6PD deficient RBCs and presumably higher predictive power of drug induced haemolytic risk.

Background Plasmodium gametocytes are sexual stages transmitted to female Anopheles mosquitoes. While Plasmodium parasites can be differentiated microscopically on Giemsa-stained blood smears, molecular methods are increasingly used because of their increased sensitivity. Molecular detection of gametocytes requires methods that discriminate between asexual and sexual stage parasites. Commonly tested gametocyte-specific mRNAs are pfs25 and pfs230 detected by reverse transcription polymerase chain reaction (RT-PCR). However, detection of these unspliced mRNA targets requires preceding DNase treatment of nucleic acids to eliminate co-purified genomic DNA. If gametocyte-specific, spliced mRNAs could be identified, DNase treatment could be eliminated and one-step multiplexed molecular methods utilized. Results Expression data was used to identify highly-expressed mRNAs in mature gametocytes that were also low in antisense RNA expression in non-gametocyte stages. After testing numerous candidate mRNAs, the spliced female Pf3D7_0630000 mRNA was selected as a Plasmodium falciparum gametocyte-specific biomarker compatible with Plasmodium 18S rRNA RT-PCR. This mRNA was only detected in samples containing mature gametocytes and was absent in those containing only asexual stage parasites or uninfected human blood. PF3D7_0630000 RT-PCR detected gametocytes across a wide range of parasite densities in both spiked and clinical samples and agreed with pfs25 RT-PCR, the gold standard for RT-PCR-based gametocyte detection. PF3D7_0630000 multiplexed with Plasmodium 18S rRNA RT-PCR was more sensitive than other spliced mRNA targets for one-step RT-PCR gametocyte detection. Conclusions Because the spliced target does not require DNase treatment, the PF3D7_0630000 assay can be multiplexed with Plasmodium 18S rRNA for direct one-step detection of gametocytes from whole human blood. Electronic supplementary material The online version of this article (doi:10.1186/s12936-017-1863-3) contains supplementary material, which is available to authorized users.