Enhancing malaria detection in resource-limited areas: A high-performance colorimetric LAMP assay for Plasmodium falciparum screening.

Malaria eradication efforts in resource-limited areas require a rapid, economical, and accurate tool for detecting of the low parasitemia. The malaria rapid diagnostic test (mRDT) is the most suitable for on-site detection of the deadliest form of malaria, Plasmodium falciparum. However, the deletions of histidine rich protein 2 and 3 genes are known to compromise the effectiveness of mRDT. One of the approaches that have been explored intensively for on-site diagnostics is the loop-mediated isothermal amplification (LAMP). LAMP is a one-step amplification that allows the detection of Plasmodium species in less than an hour. Thus, this study aims to present a new primer set to enhance the performance of a colorimetric LAMP (cLAMP) for field application. The primer binding regions were selected within the A-type of P. falciparum 18S rRNA genes, which presents a dual gene locus in the genome. The test result of the newly designed primer indicates that the optimal reaction condition for cLAMP was 30 minutes incubation at 65°C, a shorter incubation time compared to previous LAMP detection methods that typically takes 45 to 60 minutes. The limit of detection (LoD) for the cLAMP using our designed primers and laboratory-grown P. falciparum (3D7) was estimated to be 0.21 parasites/µL which was 1,000-fold higher than referencing primers. Under optimal reaction condition, the new primer sets showed the sensitivity (100%, 95% CI: 80.49-100%) and specificity (100%, 95% CI: 94.64-100%) with 100% (95% CI: 95.70-100%) accuracy on the detection of dried blood spots from Malawi (n = 84). Briefly, the newly designed primer set for P. falciparum detection exhibited high sensitivity and specificity compared to referenced primers. One great advantage of this tool is its ability to be detected by the naked eye, enhancing field approaches. Thus, this tool has the potential to be effective for accurate early parasite detection in resource-limited endemic areas.

Functional characterization of Plasmodium vivax hexose transporter 1.

Plasmodium vivax is the most widely distributed human malaria parasite. The eradication of vivax malaria remains challenging due to transmission of drug-resistant parasite and dormant liver form. Consequently, anti-malarial drugs with novel mechanisms of action are urgently demanded. Glucose uptake blocking strategy is suggested as a novel mode of action that leads to selective starvation in various species of malaria parasites. The role of hexose transporter 1 in Plasmodium species is glucose uptake, and its blocking strategies proved to successfully induce selective starvation. However, there is limited information on the glucose uptake properties via P. vivax hexose transporter 1 (PvHT1). Thus, we focused on the PvHT1 to precisely identify its properties of glucose uptake. The PvHT1 North Korean strain (PvHT1NK) expressed Xenopus laevis oocytes mediating the transport of [3H] deoxy-D-glucose (ddGlu) in an expression and incubation time-dependent manner without sodium dependency. Moreover, the PvHT1NK showed no exchange mode of glucose in efflux experiments and concentration-dependent results showed saturable kinetics following the Michaelis-Menten equation. Non-linear regression analysis revealed a Km value of 294.1 µM and a Vmax value of 1,060 pmol/oocyte/hr, and inhibition experiments showed a strong inhibitory effect by glucose, mannose, and ddGlu. Additionally, weak inhibition was observed with fructose and galactose. Comparison of amino acid sequence and tertiary structure between P. falciparum and P. vivax HT1 revealed a completely conserved residue in glucose binding pocket. This result supported that the glucose uptake properties are similar to P. falciparum, and PfHT1 inhibitor (compound 3361) works in P. vivax. These findings provide properties of glucose uptake via PvHT1NK for carbohydrate metabolism and support the approaches to vivax malaria drug development strategy targeting the PvHT1 for starving of the parasite.

Ring stage classification of Babesia microti and Plasmodium falciparum using optical diffraction 3D tomographic technique.

BACKGROUND: Babesia is an intraerythrocytic parasite often misdiagnosed as a malaria parasite, leading to inappropriate treatment of the disease especially in co-endemic areas. In recent years, optical diffraction tomography (ODT) has shown great potential in the field of pathogen detection by quantification of three-dimensional (3D) imaging tomograms. The 3D imaging of biological cells is crucial to investigate and provide valuable information about the mechanisms behind the pathophysiology of cells and tissues. METHODS: The early ring stage of P. falciparum were obtained from stored stock of infected RBCs and of B. microti were obtained from infected patients during diagnosis. The ODT technique was applied to analyze and characterize detailed differences between P. falciparum and B. microti ring stage at the single cell level. Based on 3D quantitative information, accurate measurement was performed of morphological, biochemical, and biophysical parameters. RESULTS: Accurate measurements of morphological parameters indicated that the host cell surface area at the ring stage in B. microti was significantly smaller (140.2 ± 17.1 µm2) than that in P. falciparum (159.0 ± 15.2 µm2), and sphericities showed higher levels in B. microti-parasitized cells (0.66 ± 0.05) than in P. falciparum (0.60 ± 0.04). Based on biochemical parameters, host cell hemoglobin level was significantly higher and membrane fluctuations were respectively more active in P. falciparum-infected cells (30.25 ± 2.96 pg; 141.3 ± 24.68 nm) than in B. microti (27.28 ± 3.52 pg; 110.1 ± 38.83 nm). The result indicates that P. falciparum more actively altered host RBCs than B. microti. CONCLUSION: Although P. falciparum and B. microti often show confusable characteristics under the microscope, and the actual three-dimensional properties are different. These differences could be used in differential clinical diagnosis of erythrocytes infected with B. microti and P. falciparum.

The impact of Anopheles gambiae egg storage for mass rearing and production success.

BACKGROUND: Mass rearing requires a large colony from which male individuals can be harvested for sterilization and release. Attention is needed when monitoring life parameters of the reared population, knowing that any variations within the target population would lead to mismatching between two populations. The aim of this study was to assess the impact of Anopheles gambiae sensu stricto (s.s.) egg storage on hatchability and life history traits. For each parameter, comparison was made between freshly laid and stored eggs in three densities (40, 80, 120 eggs). METHODS: Anopheles gambiae s.s. freshly laid eggs were collected from the Tropical Pesticide Research Institute (TPRI) insectary. Eggs to be stored were kept at - 20 °C for 10 min and then transferred to refrigerators at 4 °C for intervals of 5, 10, 15, 20, and 25 days. After respective storage days, the eggs were transferred from refrigerators to ambient temperature of (25 ± 2) °C for 24 h and then placed in incubators for 24 h. Thereafter eggs were hatched. The egg hatchability, emerged larvae development, larvae survival and emerged adult sex ratios were monitored. RESULTS: This study found that hatching rates decreased with increase in storage time. The difference was significant in eggs stored for 10 and 15 days (P < 0.05). There were no significant differences in hatching rates between An. gambiae eggs stored for 5 days and freshly hatched eggs (P > 0.05). Anopheles larvae development (L1 to pupae) was not significantly affected by storage time across all hatching densities. The study also found that larvae survival decreased with increase in egg storage time. However, there was no significant difference between larvae from freshly hatched eggs and those from eggs at 5 and 10 storage days (P > 0.05) but not for eggs stored for 15 days. Furthermore, there was a decrease in emerged adult males and increase in females relative to increased time of egg storage. The difference was significant (P < 0.05) at 15 storage days but not for eggs stored for 5 and 10 days (in triplicate densities). CONCLUSION: From this study it was concluded that storing An. gambiae eggs at 4 °C and 48 ± 2% relative humidity (RH) for 5 days is the optimal condition and time that did not affect egg hatching rates, larval development and survivorship and emerged adult mosquito sex ratio.