Plasmodium culture in vitro is often used as an antimalarial drug evaluation model, but the lifecycle of P. falciparum culture in vitro tends to be disordered, which affects the research and evaluation of antimalarial drug mechanism in vitro. By combining magnetic bead separation method with sorbitol synchronization method, a synchronization method was constructed to quickly acquire different lifecycles of P. falciparum and obtain large amounts of parasite with a narrow synchronization window in a short period. Furthermore, the dihydroartemisinin(DHA) was used to treat the early trophozoite phase of P. falciparum 3 D7 for 4 h. Then mRNA was extracted and RNA-seq was conducted to analyze the differential expression of mRNA after drug treatment and obtain the differential gene expression profile. Differential expression of up-regulated genes and down-regulated genes was analyzed according to the screening criteria of |log_2FC|>1 and P<0.05. There, 262 genes were up-regulated and 77 genes were down-regulated. GO functional enrichment analysis of all the differentially expressed genes showed that the enrichment items mainly included cell membrane components, transporter activity, serine/threonine kinase activity, Maurer's clefts(MCs), rhoptry, antigen variation and immune evasion. The enrichment of KEGG pathway included malaria, fatty acid metabolism and peroxisome. Protein-protein interaction(PPI) analysis showed that the down-regulated genes in the modules with high degree of association included rhoptry, myosin complex, transporter and other genes related to the important life activities of malaria invasion and immune escape; the up-regulated genes were mainly related to various toxic exportins of malaria, such as PfSBP1 of MCs. qRT-PCR was used to verify the expression level of some genes, and most of the results were the same as the sequencing results. SBP1 was significantly up-regulated, while some antigenic protein expression levels were down-regulated. Above all, key molecules of DHA therapy were mainly involved in the parasites' rhoptry, transporter, antigenic variation, plasmodium exportin. These results offer us many hints to guide the further studies on mechanism of artemisinin and provide a new way for development of new antimalarial drugs.
Animals ; Antimalarials ; Artemisinins ; Erythrocytes ; Plasmodium falciparum ; Transcriptome

Detailed description of malaria in low transmission areas is crucial for elimination. The current study aimed to provide a comprehensive description for malaria transmission in Jazan, a low transmission district, southwestern Saudi Arabia. Patients at a tertiary care hospital were recruited in our study between August 2016 and September 2018. Malaria diagnosis was performed through a species-specific nested polymerase chain reaction (nested PCR), microscopy and Paramax-3™ rapid detection test (RDT). Malaria was detected in 30 patients by the PCR, with point prevalence of 10.9%. Of these malaria infections, 80% was imported, 26.6% was asymptomatic and 23.3% was sub-microscopic. Malaria was reported throughout the year, with February/March and September/October peaks. Infection was significantly more in males than in females (P=0.01). Likewise, infections were detected more in febrile than in non-febrile patients (P=0.01). Adult aged 15–24 years, fever and travel were identified as high-risk factors. Malaria was primarily attributed to Plasmodium falciparum mono-infections, followed by P. vivax mono-infections and lastly to falciparum/vivax mixed infections accounting 76.6%, 16.6%, and 6.6% of PCR-confirmed malaria cases, respectively. The nested PCR was superior to the smear microscopy (sensitivity 76.6%; specificity 100%) and the RDT (sensitivity 83.3%, specificity 94.2%). The overall percent agreement between microscopy and the RDT was 92.7% (kappa=0.63). High proportion of imported malaria including sub-microscopic and sub-patent cases were described. We suggest that incorporation of molecular tool into the conventional malaria diagnosis is beneficial in Jazan district.
Adult ; Coinfection ; Diagnosis ; Female ; Fever ; Humans ; Malaria ; Male ; Microscopy ; Plasmodium falciparum ; Polymerase Chain Reaction ; Prevalence ; Saudi Arabia ; Sensitivity and Specificity ; Tertiary Healthcare

Artemisinin-based combination therapy (ACT) resistance is widespread throughout the Greater Mekong Subregion. This raises concern over the antimalarial treatment in Thailand since it shares borders with Cambodia, Laos, and Myanmar where high ACT failure rates were reported. It is crucial to have information about the spread of ACT resistance for efficient planning and treatment. This study was to identify the molecular markers for antimalarial drug resistance: Pfkelch13 and Pfmdr1 mutations from 5 provinces of southern Thailand, from 2012 to 2017, of which 2 provinces on the Thai- Myanmar border (Chumphon and Ranong), one on Thai-Malaysia border (Yala) and 2 from non-border provinces (Phang Nga and Surat Thani). The results showed that C580Y mutation of Pfkelch13 was found mainly in the province on the Thai-Myanmar border. No mutations in the PfKelch13 gene were found in Surat Thani and Yala. The Pfmdr1 gene isolated from the Thai-Malaysia border was a different pattern from those found in other areas (100% N86Y) whereas wild type strain was present in Phang Nga. Our study indicated that the molecular markers of artemisinin resistance were spread in the provinces bordering along the Thai-Myanmar, and the pattern of Pfmdr1 mutations from the areas along the international border of Thailand differed from those of the non-border provinces. The information of the molecular markers from this study highlighted the recent spread of artemisinin resistant parasites from the endemic area, and the data will be useful for optimizing antimalarial treatment based on regional differences.
Cambodia ; Drug Resistance ; Laos ; Myanmar ; Parasites ; Plasmodium falciparum ; Plasmodium ; Thailand

Indonesia and South Korea have become inseparable in various respects since the 2 countries established diplomatic relation in 1973. Indonesia is a tropical region that stretches across the equator, comprised of 5 main islands (Java, Kalimantan, Sumatra, Sulawesi, and Papua) and 4 archipelagoes (Riau, Bangka Belitung, Nusa Tenggara, and Maluku). As most population of Eastern Indonesia (Sulawesi, Papua and Nusa Tenggara & Maluku) live in poor areas, it is expected that there will be many parasites. Nevertheless, little is known about the status of parasites in Indonesia. This study examines the prevalences of malaria and lymphatic filaria, which are prevalent in Indonesia, as well as those of soil-transmitted-helminths (STH). As a result, the Plasmodium falciparum and P. vivax case loads are almost equal. The current prevalence of P. vivax is uniformly low (<5%) in all age groups and annual parasite incidence (API) showed decreasing tendency as 0.84 per 1,000 population in 2016. However, more than 65 million people still live in malaria epidemic regions. Lymphatic filariasis remains an important public health problem and 236 cities were classified as endemic areas in 514 cities/districts in 2017. It is difficult to ascertain the current prevalence rate of STH in Indonesia, although West Sumba and Southwest Sumba in East Nusa Tenggara reported prevalence rate of more than 20%. The study also considers the (sero) prevalences of other parasites identified in Indonesia. This report should be useful not only to parasitologists but also to travelers and people with business in Indonesia.
Commerce ; Diplomacy ; Elephantiasis, Filarial ; Humans ; Incidence ; Indonesia ; Islands ; Korea ; Malaria ; Parasites ; Plasmodium falciparum ; Prevalence ; Public Health

Mixed-species malaria infections are often unrecognized or underestimated. We hereby report the first described case of mixed infection with Plasmodium falciparum and Plasmodium ovale malaria in a returned traveller in Korea. In August 2016, a 25-year-old returned traveller from Cameroon and Democratic Republic of Congo presented with fever. He was diagnosed as P. falciparum malaria and successfully treated with artesunate. And 5 weeks after the completion of treatment, he presented with fever and diagnosed as P. ovale infection. P. ovale infection is a rare cause of malaria and often shows delayed presentation due to its dormant liver stage as hypnozoites. At re-presentation, the immunochromatographic test and microscopic examinations of our patient did not reveal P. ovale, which was only detected via polymerase chain reaction (PCR) assay. This case highlights the importance of considering malaria infection even in persons who have previously received malaria treatment. It also shows the usefulness of PCR testing for diagnosing P. ovale infections, which often present with a low level of parasitaemia.
Adult ; Cameroon ; Coinfection* ; Congo ; Fever ; Humans ; Korea* ; Liver ; Malaria ; Plasmodium falciparum* ; Plasmodium ovale* ; Plasmodium* ; Polymerase Chain Reaction

Molecular techniques have been introduced for malaria diagnosis because they offer greater sensitivity and specificity than microscopic examinations. Therefore, DNA isolation methods have been developed for easy preparation and cost effectiveness. The present study described a simple protocol for Plasmodium DNA isolation from EDTA-whole blood. This study demonstrated that after heating infected blood samples with Tris–EDTA buffer and proteinase K solution, without isolation and purification steps, the supernatant can be used as a DNA template for amplification by PCR. The sensitivity of the extracted DNA of Plasmodium falciparum and Plasmodium vivax was separately analyzed by both PCR and semi-nested PCR (Sn-PCR). The results revealed that for PCR the limit of detection was 40 parasites/μl for P. falciparum and 35.2 parasites/μl for P. vivax, whereas for Sn-PCR the limit of detection was 1.6 parasites/μl for P. falciparum and 1.4 parasites/μl for P. vivax. This new method was then verified by DNA extraction of whole blood from 11 asymptomatic Myanmar migrant workers and analyzed by Sn-PCR. The results revealed that DNA can be extracted from all samples, and there were 2 positive samples for Plasmodium (P. falciparum and P. vivax). Therefore, the protocol can be an alternative method for DNA extraction in laboratories with limited resources and a lack of trained technicians for malaria diagnosis. In addition, this protocol can be applied for subclinical cases, and this will be helpful for epidemiology and control.
Cost-Benefit Analysis ; Diagnosis ; DNA ; Edetic Acid ; Endopeptidase K ; Epidemiology ; Heating ; Hot Temperature ; Humans ; Limit of Detection ; Malaria ; Methods ; Myanmar ; Plasmodium falciparum ; Plasmodium vivax ; Plasmodium ; Polymerase Chain Reaction ; Sensitivity and Specificity ; Transients and Migrants

Development of an effective vaccine is critically needed for the prevention of malaria. One of the key antigens for malaria vaccines is the apical membrane antigen 1 (AMA-1) of the human malaria parasite Plasmodium falciparum, the surface protein for erythrocyte invasion of the parasite. The gene encoding AMA-1 has been sequenced from populations of P. falciparum worldwide, but the haplotype diversity of the gene in P. falciparum populations in the Greater Mekong Subregion (GMS), including Thailand, remains to be characterized. In the present study, the AMA-1 gene was PCR amplified and sequenced from the genomic DNA of 65 P. falciparum isolates from 5 endemic areas in Thailand. The nearly full-length 1,848 nucleotide sequence of AMA-1 was subjected to molecular analyses, including nucleotide sequence diversity, haplotype diversity and deduced amino acid sequence diversity and neutrality tests. Phylogenetic analysis and pairwise population differentiation (F( st) indices) were performed to infer the population structure. The analyses identified 60 single nucleotide polymorphic loci, predominately located in domain I of AMA-1. A total of 31 unique AMA-1 haplotypes were identified, which included 11 novel ones. The phylogenetic tree of the AMA-1 haplotypes revealed multiple clades of AMA-1, each of which contained parasites of multiple geographical origins, consistent with the F(st) indices indicating genetic homogeneity or gene flow among geographically distinct populations of P. falciparum in Thailand's borders with Myanmar, Laos and Cambodia. In summary, the study revealed novel haplotypes and population structure needed for the further advancement of AMA-1-based malaria vaccines in the GMS.
Amino Acid Sequence ; Base Sequence ; Cambodia ; DNA ; Erythrocytes ; Gene Flow ; Haplotypes ; Humans ; Laos ; Malaria ; Malaria Vaccines ; Membranes ; Myanmar ; Parasites ; Plasmodium falciparum ; Plasmodium ; Polymerase Chain Reaction ; Polymorphism, Genetic ; Thailand ; Trees

The incidence of vector-borne diseases has increased with climate change and developments of transportation. Malaria was eliminated in the early 1980s in Korea, but one domestic case was reported in 1993. The number of patients increased to 4,142 in 2002, and less than 700 cases of malaria are reported annually nowadays. Additionally, approximately 70 to 80 imported malaria cases were reported annually, and most of them are Plasmodium falciparum infections from Africa. Annually, 20 to 40 cases of Japanese encephalitis are reported, and the incidence is high in individuals between the ages of 40 and 59. Moreover, 313 cases of dengue fever were imported in 2016, and most of them were from Southeast Asia. In 2016, 11,000 cases of scrub typhus were reported, and most of the patients were older than 50. The number of reported cases of severe fever with thrombocytopenia syndrome has been increasing since 2013, mostly in patients older than 60. It is important to avoid vectors for the prevention, and for the doctors to diagnose the apatients as soon as possible.
Africa ; Asia, Southeastern ; Climate Change ; Dengue ; Encephalitis, Japanese ; Fever ; Humans ; Incidence ; Korea ; Malaria ; Plasmodium falciparum ; Scrub Typhus ; Thrombocytopenia ; Transportation

Blackwater fever is a serious clinical syndrome manifested by acute intravascular hemolysis, fever, and the passage of black or red urine, which is classically associated with falciparum malaria and irregular administration of quinine. In Korea, Plasmodium vivax is the only endemic malaria circulating; a number of imported cases of falciparum malaria have been reported in patients following return from international travel to a malaria endemic area. Therefore, it is important for health care professionals including pediatricians to be aware of the falciparum malaria and its clinical courses. Herein, we report a case of a 14-year-old girl with severe falciparum malaria that was complicated by blackwater fever.
Adolescent ; Blackwater Fever* ; Child* ; Delivery of Health Care ; Female ; Fever ; Hemolysis ; Humans ; Korea ; Malaria* ; Malaria, Falciparum ; Plasmodium vivax ; Quinine

The drug-resistance of malaria parasites is the main problem in the disease control. The huge Brazilian biodiversity promotes the search for new compounds, where the animal kingdom is proving to be a promising source of bioactive compounds. The main objective of this study was to evaluate the antiplasmodial and cytotoxic activity of the compounds obtained from the toad venoms of Brazilian Amazon. Toad venoms were collected from the secretion of Rhinella marina and Rhaebo guttatus in Mato Grosso State, Brazil. The powder was extracted at room temperature, yielding 2 extracts (RG and RM) and a substance ('1') identified as a bufadienolide, named telocinobufagin. Growth inhibition, intraerythrocytic development, and parasite morphology were evaluated in culture by microscopic observations of Giemsa-stained thin blood films. Cytotoxicity was determined against HepG2 and BGM cells by MTT and neutral red assays. The 2 extracts and the pure substance ('1') tested were active against chloroquine-resistant Plasmodium falciparum strain, demonstrating lower IC₅₀ values. In cytotoxic tests, the 2 extracts and substance '1' showed pronounced lethal effects on chloroquine-resistant P. faciparum strain and low cytotoxic effect, highlighting toad parotoid gland secretions as a promising source of novel lead antiplasmodial compounds.
Amphibian Venoms* ; Animals ; Biodiversity ; Brazil* ; Bufo marinus ; Malaria ; Neutral Red ; Parasites ; Plasmodium falciparum