A novel peptide pair-based rapid fluorescent diagnostic system for malaria Plasmodium falciparum detection.

Advanced diagnostic materials, such as aptamers, are required due to the scarcity of efficient diagnostic antibodies and the low sensitivity of rapid diagnostic kits at detecting the malaria parasite, Plasmodium falciparum. METHODS: Two peptides M2.9 [(KPTAEQTESPELQSAPEN) and M2.17 (KILFNVYSPLGCTCECWV)] were designed using simple epitope prediction tools and modified against the merozoite surface antigen 2 of P. falciparum (Pf.MSP2) by 3-dimensional modeling based on binding affinity. Based on five prediction tools for hydropathy, M2.17 was selected as an appropriate capture peptide. A peptide-based fluorescence-linked immunosorbent assay (FLISA) and a peptide pair-based fluorescent immunochromatographic test strip (FICT) were developed to detect P. falciparum 3D7 (drug-sensitive) and P. falciparum K1 (multi drugs-resistant) strains. RESULTS: Bioinformatic analysis of two peptides demonstrated the potential binding affinity with the merozoite surface protein 2 of P. falciparum (Pf.MSP2) with a positive hydropathy value. The limit of detection (LOD) of FLISA was 10 parasites/µL and of a peptide pair-linked rapid FICT system was 5 and 200 parasites/µL for P. falciparum 3D7 and K1, respectively. Compared to commercial rapid detection systems (RDTs), a peptide pair-linked FICT system exhibited a 20-fold greater efficiency in detecting P. falciparum 3D7 and specifically discriminated another protozoan spp. CONCLUSION: A peptide pair-linked rapid diagnostic strip could be an alternative to conventional RDTs for monitoring wild-type and drug-resistant malaria parasites.

Evaluation of the antimalarial activity of SAM13-2HCl with morpholine amide (SKM13 derivative) against antimalarial drug-resistant Plasmodium falciparum and Plasmodium berghei infected ICR mice.

Antimalarial drugs are an urgently need and crucial tool in the campaign against malaria, which can threaten public health. In this study, we examined the cytotoxicity of the 9 antimalarial compounds chemically synthesized using SKM13-2HCl. Except for SKM13-2HCl, the 5 newly synthesized compounds had a 50% cytotoxic concentration (CC50) > 100 µM, indicating that they would be less cytotoxic than SKM13-2HCl. Among the 5 compounds, only SAM13-2HCl outperformed SKM13-2HCl for antimalarial activity, showing a 3- and 1.3-fold greater selective index (SI) (CC50/IC50) than SKM13-2HCl in vitro against both chloroquine-sensitive (3D7) and chloroquine -resistant (K1) Plasmodium falciparum strains, respectively. Thus, the presence of morpholine amide may help to effectively suppress human-infectious P. falciparum parasites. However, the antimalarial activity of SAM13-2HCl was inferior to that of the SKM13-2HCl template compound in the P. berghei NK65-infected mouse model, possibly because SAM13-2HCl had a lower polarity and less efficient pharmacokinetics than SKM13-2HCl. SAM13-2HCl was more toxic in the rodent model. Consequently, SAM13-2HCl containing morpholine was selected from screening a combination of pharmacologically significant structures as being the most effective in vitro against human-infectious P. falciparum but was less efficient in vivo in a P. berghei-infected animal model when compared with SKM13-2HCl. Therefore, SAM13-2HCl containing morpholine could be considered a promising compound to treat chloroquine-resistant P. falciparum infections, although further optimization is crucial to maintain antimalarial activity while reducing toxicity in animals.

Identification of breeding habitats and kdr mutations in Anopheles spp. in South Korea.

BACKGROUND: Malaria is still endemic in South Korea. However, limited information is available on the current Anopheles breeding sites and the occurrence of insecticide resistance-associated genetic mutations and their distribution needed to control the malaria vector efficiently. METHODS: This study explored breeding sites of Anopheline adults in Gimpo-si, near the demilitarized zone (DMZ) in Gyeonggi-do province, South Korea, from 2022 to 2023. Genetic diversity was investigated based on the internal transcribed spacer (ITS2), cytochrome c oxidase subunit I (COI), and knockdown resistance (kdr) genes of Anopheles mosquitoes. A natural environment associated with the seasonal abundance of Anopheles larvae was characterized. RESULTS: Two breeding sites of Anopheles larvae and adults were found at a stream margin or shallow freshwater near the forest in Wolgot-myeon in Gimpo-si without cattle shed within 1 km and in Naega-myeon in Ganghwa-gun with cow shed within 100 m in 2022 and 2023, respectively. Both sites were located between the newly cultivated lands and the forest. Besides, both breeding sites were in the valley at a slight elevation of 60-70 m from ground lands and maintained the shadow all day. Overall, the Wolgot-myeon breeding site showed various Anopheles spp. larvae, including Anopheles sinensis. Naega-myeon, an additional breeding site found in 2023, had Anopheles sineroides larvae, and approximately 59.7% (89/149) of An. sinensis adults inhabited within a 100-m distance. The total collection, including larvae and adults, revealed that An. sinensis, Anopheles pullus, Anopheles kleini, An. sineroides, Anopheles belenrae, and Anopheles lindesayi accounted for 44.2% (118/267), 0.7% (2/267), 0.7% (2/267), 22.1% (59/267), 1.9% (5/267), and 30.3% (81/267), respectively. Furthermore, various kdr mutant genotypes (F/F, C/C, L/F, L/C and F/C) in An. sinensis, and the first kdr allele mutant (L/F1014) in An. belenrae were identified in South Korea. CONCLUSIONS: Two breeding sites of Anopheles larvae were studied in Wolgot-myeon and Naega-myeon. Various Anopheles spp. larvae were detected in both habitats, but overall, An. sinensis was the most prevalent adults in both study sites. The occurrence of kdr allele mutant of An. belenrae in South Korea was reported. Rigorous larvae monitoring of Anopheles spp., continuously updating information on Anopheles breeding sites, and understanding the environmental conditions of Anopheles habitats are required to develop an effective malaria control programme in South Korea.