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Background Protection against Plasmodium falciparum is observed in a population deficient in glucose-6-phosphate dehydrogenase (G6PD), particularly in African and Mediterranean regions. However, such protection remains unknown among G6PD-deficient individuals in Southeast Asia. Methods In this study, we assessed the invasion and maturation of P falciparum K1 in a culture of erythrocytes isolated from Thai subjects carrying Viangchan (871G > A) and Mahidol (487G > A). Results We found that the parasites lost their ability to invade hemizygous and homozygous G6PD-deficient erythrocytes of Viangchan and Mahidol variants in the second and third cycles of intraerythrocytic development. It is interesting to note that P falciparum parasites selectively grew in erythrocytes from hemi- and homozygous genotypes with normal G6PD activity. Moreover, externalization of phosphatidylserine upon P falciparum infection was significantly increased only in Viangchan hemizygous variant cells. Conclusions This study is the first to show that blockage of invasion in long-term culture and potentially enhanced removal of parasitized erythrocytes were observed for the first time in erythrocytes from Viangchan and Mahidol G6PD-deficient individuals.

Background Extracellular vesicles (EVs) have been broadly studied in malaria for nearly a decade. These vesicles carry various functional biomolecules including RNA families such as microRNAs (miRNA). These EVs-derived microRNAs have numerous roles in host-parasite interactions and are considered promising biomarkers for disease severity. However, this field lacks clinical studies of malaria-infected samples. In this study, EV specific miRNAs were isolated from the plasma of patients from Thailand infected with Plasmodium vivax and Plasmodium falciparum. In addition, it is postulated that these miRNAs were differentially expressed in these groups of patients and had a role in disease onset through the regulation of specific target genes. Methods EVs were purified from the plasma of Thai P. vivax-infected patients (n = 19), P. falciparum-infected patients (n = 18) and uninfected individuals (n = 20). EV-derived miRNAs were then prepared and abundance of hsa-miR-15b-5p, hsa-miR-16-5p, hsa-let-7a-5p and hsa-miR-150-5p was assessed in these samples. Quantitative polymerase chain reaction was performed, and relative expression of each miRNA was calculated using hsa-miR-451a as endogenous control. Then, the targets of up-regulated miRNAs and relevant pathways were predicted by using bioinformatics. Receiver Operating Characteristic with Area under the Curve (AUC) was then calculated to assess their diagnostic potential. Results The relative expression of hsa-miR-150-5p and hsa-miR-15b-5p was higher in P. vivax-infected patients compared to uninfected individuals, but hsa-let-7a-5p was up-regulated in both P. vivax-infected patients and P. falciparum-infected patients. Bioinformatic analysis revealed that these miRNAs might regulate genes involved in the malaria pathway including the adherens junction and the transforming growth factor-β pathways. All up-regulated miRNAs could potentially be used as disease biomarkers as determined by AUC; however, the sensitivity and specificity require further investigation. Conclusion An upregulation of hsa-miR-150-5p and hsa-miR-15b-5p was observed in P. vivax-infected patients while hsa-let-7a-5p was up-regulated in both P. vivax-infected and P. falciparum-infected patients. These findings will require further validation in larger cohort groups of malaria patients to fully understand the contribution of these EVs miRNAs to malaria detection and biology.

Background Gold standard microscopic examination of Plasmodium falciparum intraerythrocytic stage remains an important process for staging and enumerating parasitized erythrocytes in culture; however, microscopy is laborious and its accuracy is dependent upon the skill of the examiner. Methods In this study, ViSafe Green (VSG), which is a nucleic acid-binding fluorescent dye, was used for assessing in vitro development of P. falciparum using flow cytometry. Results Fluorescence intensity of VSG was found to depend on the developmental stage of parasites. Specifically, multiple-nuclei-containing schizonts were observed in the VSGhigh population, and growing trophozoites and ring-shaped forms were observed in the VSGintermediate and VSGlow populations. The efficacy of VSG-based assay was found to be comparable to the microscopic examination method, and it demonstrated an ability to detect as low as 0.001% of the parasitaemia estimated by Giemsa staining. Moreover, when applying VSG for anti-malarial drug test, it was able to observe the growth inhibitory effect of dihydroartemisinin, the front-line drug for malaria therapy. Conclusions Taken together, the results of this study suggest the VSG-based flow cytometric assay to be a simple and reliable assay for assessing P. falciparum malaria development in vitro.

Additional file 2: Fig. S2. VSG-based flow cytometric analysis of gametocyte. Early schizonts and mature schizonts (left panel) and early stage gametocytes (right panel) were observed in VSGhigh fraction. In the VSGhigh fraction, parasitized erythrocytes have granular distribution of haemozoin, resembling stage IB. Moreover, some were elongated and D-shaped within erythrocytes, which are key characteristics of stage II gametocytes. Early schizonts having 2 and 6 divided nucleus, and mature schizonts consisting of 14 merozoites were also observed in the VSGhigh fraction, whereas ring forms and trophozoites were observed in the VSGlow and VSGintermediate fractions. Scale bar: 10 μm.

Additional file 3: Fig. S3. Giemsa stain of VSG+ cells sorted from non-synchronized and synchronized culture of P. falciparum. (A) Morphology of three distinct populations observed in the non-synchronized culture (Fig. 5b, left panel): (1) cells having small size with various granularity (approximately 0–45 K of FSC-A, and 30–170 K of SSC-A); (2) cells having a relatively large size with high granularity (approximately 45–185 K of FSC-A, and 75–170 K of SSC-A); and, (3) cells having a relatively larger size with low granularity (approximately 45–185 K of FSC-A, and 20–75 K of SSC-A). Based on the intensity of VSG and microscopic images, the population number 1, 2, and 3 in the upper panel of Fig. 5b are schizonts, trophozoites, and ring forms, respectively. (B) Morphology of one minor (indicated as 1) and one major (indicated as 2) population observed in the synchronized culture (Fig. 5b, right panel): (1) cells having high granularity (more than 160 K of SSC-A); and (2) cells having with low granularity (lower than 160 K of SSC-A). Both had a similar size (50–150 K of FSC-A). Based on microscopic images, majority of the VSG+ cells with more than 160-K SSC-A are infected erythrocytes containing multiple ring forms, and they had VSG intensity of 11,578; whereas, the majority of VSG+ cells with lower than 160 K SSC-A are infected erythrocytes with single ring forms and they had VSG intensity of 10,182.

Additional file 1: Fig. S1. Effect of cell debris on analysis of VSG+ cells by flow cytometry. (A) In flow cytometry, cell debris has a lower level of forward scatter (FSC), and it can be observed at the bottom left corner of the density plot [32]. To remove cell debris, the threshold of FSC was set at 10,000 for all experiments. The presence of debris in three independent experiments was examined and shown. Cells having characteristics of FSClow and that are located at bottom left (circles) of the density plot are considered debris. There is 0.64±0.2% of debris, suggesting that the majority of analysed cells are not debris. Moreover, the presence of cell debris in VSG+ cells was checked by examining VSG+ cells according to FSC-A and SSC-A (lower panels). There was 0.69±0.13% of VSG+ cells that had characteristics of FSClow at bottom left corner of the density plot. Therefore, more than 95% of cells analysed by VSG-based flow cytometry were unlikely to be cell debris. (B) Morphology of cells having characteristics of FSClow (< 50 K). P. falciparum-infected erythrocytes were subjected to flow cytometric analysis. The gated single cells were sorted according to intensity of FSC. There were ring forms and schizonts observed in the FSClow cells. Thus, inclusion of the FSClow cells is required. Microscopic images were captured using a microscope with objective lens of 100x magnification.