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The use of an effective antimalarial drug is the cornerstone of malaria control. However, the development and spread of resistant Plasmodium falciparum strains have placed the global eradication of malaria in serious jeopardy. Molecular marker analysis constitutes the hallmark of the monitoring of Plasmodium drug-resistance. This study included 96 P. falciparum PCR-positive samples from southern Somalia. The P. falciparum chloroquine resistance transporter gene had high frequencies of K76T, A220S, Q271E, N326S, and R371I point mutations. The N86Y and Y184F mutant alleles of the P. falciparum multidrug resistance 1 gene were present in 84.7 and 62.4% of the isolates, respectively. No mutation was found in the P. falciparum Kelch-13 gene. This study revealed that chloroquine resistance markers are present at high frequencies, while the parasite remains sensitive to artemisinin (ART). The continuous monitoring of ART-resistant markers and in vitro susceptibility testing are strongly recommended to track resistant strains in real time.

This paper presents synthesis of novel decorated isoquinolines that can be used as antimalarial agents. Two series of compounds, isoquinoline phenyl and isoquinoline triazole derivatives, were synthesized via the Suzuki–Miyaura and Sharpless–Fokin reactions respectively. The final compounds were investigated for their antimalarial activity in cell-based experiments. In the series of isoquinoline phenyl derivatives, compound 6 exhibited interesting antiplasmodial activity against chloroquine resistant (K1) and chloroquine sensitive (3D7) strains of P. Falciparum with IC50 1.91 ± 0.21 μM and 2.31 ± 0.33 μM, respectively. In the series of isoquinoline-triazole derivatives, compound 15 was the most active against resistant (K1) and sensitive (3D7) strains of P. falciparum with IC50 4.55 ± 0.10 μM and 36.91 ± 2.83 μM, respectively. The respective resistance indices of compounds 6 and 15 against K1 and 3D7 were 0.83 and 0.12. Compound 15 was promisingly effective against the resistant strain. The results of this study provided useful contributions for further lead discovery and lead modification of modern rational drug design.

Backgroundobjectives: Changes in parasite biology, particularly the gametocytogenesis process, could be one of the important contributing factors for worldwide malaria resurgence. The present study investigated the prevalence rates of pretreatment gametocyte carriage and density in Plasmodium falciparum and P. vivax infections in the low malaria-endemic area on the Thai-Myanmar border.One hundred and twenty-six blood samples were collected from patients with signs and symptoms of malaria who attended malaria clinics. Malaria positive cases detected by microscopic examination were confirmed by species-specific nested-PCR in 97 (29 and 68 samples for P. falciparum and P. vivax, respectively).The proportion of P. vivax and P. falciparum-infected samples was 70.1: 29.9%. The density in P. falciparum positive samples [median (95%CI): 10,340 (5280-19,200) μ/l] was significantly higher than P. vivax positive samples [4508 (3240-6120) μ/l]. Sixteen out of twenty-nine (55.2%) and 36 out of 68 (52.9%) P. falciparum- and P. vivax-infected samples, respectively, were gametocyte-positive. Gametocyte density in the P. falciparum-infected[124 (69-253) /μl] was significantly higher than that of the P. vivax-infected [54 (45-70)/μl] samples. A significant correlation between gametocyte density and pretreatment parasitemia was only detected in P. falciparum-infected, but not P. vivax-infected samples.The observed high prevalence rates of pretreatment gametocyte carriage of both malaria species, which serves as a large malaria reservoir, particularly in P. falciparum infection, could have a significant impact on malaria control in the endemic populations.

Objective The study aimed to investigate the inhibitory effects of AL on the ERK signaling molecules (ERK, p-ERK, cyclin D, and eIF4B) and the growth and proliferation of CCA cells. Materials and methods The viability of the three CCA cell lines CL-6, HuCCT1, and HuH28 was determined using MTT assay. The effect of Ras/ERK inhibitors on protein expression in the presence of AL extract was investigated. The protein extracted from each CCA cell following exposure to AL and/or Ras/ERK inhibitors were separated on 12.5% SDS-PAGE. The analysis of mRNA expression following 48 and 72 hours of AL exposure in comparison with 0 hours (non-exposed cells) was performed by using RT-PCR. Results The potency of cytotoxic activity of AL (by MTT assay) was about three times higher than the standard drug 5-fluorouracil. The IC50 (concentration that inhibits cell growth by 50%) of AL for the CL-6, HuCCT-1 and HuH28 cell lines were 29.77±6.64, 35.45±4.96, and 35.32±6.69 µg/mL (mean+SD), respectively. The cells were exposed to AL extract at the IC50 for 0, 12, 24, 48, and 72 hours in the absence and presence of Ras/ERK inhibitors (salirasib and XMD8-92). Protein expression was determined by Western blot analysis. The results suggested the lack of significant inhibitory effect of AL on ERK at 48 and 72 hours of exposure in all CCA cell types. On the other hand, a significant inhibitory effect was observed with p-ERK expression in all CCA cell types. Cyclin D was significantly down-regulated at 72 hours of exposure in all cell types with different potencies. The expression of eIF4B was markedly inhibited in HuCCT-1 but slightly inhibited in CL-6 and HuH28 cells. Real-time PCR analysis revealed significant down-regulation of ERK following 72 hours of AL exposure in the HuCCT1 and HuH28, but not CL-6 cell. Conclusion The ERK signaling cascade and downstream molecules are potential targets of action of AL in CCA.

Purpose The purpose of this paper is to investigate epidemiology and control strategies of the four priority vector-borne diseases (VBDs) in Nepal, i.e. malaria, Kala-azar (visceral leishmaniasis), lymphatic filariasis (LF) and dengue fever/dengue hemorrhagic fever. Design/methodology/approach The study was a retrospective design to collect data during 1998–2016 from VBDs endemic districts of Nepal. All data were reviewed and epidemiological information of the four VBDs were analyzed. Findings The number of malaria cases during 1998–2016 of the 13 affected districts was declined from 8,498 to 991 cases with no record of deaths since 2012. The number of cases and deaths in the 12 kala-azar (visceral leishmaniasis) affected districts in 1998 was 1,409 and 42 cases, respectively, but was dramatically decreased in 2016 to 213 and 2 cases, respectively. LF cases of the 61 affected districts in 2011, 2014 and 2016 were 28,855, 30,000 and 33,517 cases, respectively. In total, 25 districts achieved elimination target and the remaining are expected to complete the needful cycles by 2018. Dengue incidence of the 31 affected districts during 2006–2015 was under controlled with reported cases of 642, 356 and 136 cases in 2013, 2014 and 2015, respectively, and only one death in 2015. Implementation of control strategies particularly disease management and community peoples’ awareness significantly reduced the cases and deaths of the target VBDs. Practical implications The results of this study clearly suggest that the current control strategies have been worked effectively. However, in particular of the VBDs, health education in communities in the endemic areas should be adopted for better community participation in the context of the primary health care approach and increase the effectiveness of disease control. Originality/value VBDs, i.e., malaria, kala-azar (visceral leishmaniasis), LF and dengue fever/dengue hemorrhagic fever, are major causes of morbidity and mortality in the least developed countries which include Nepal. Globalization of travel and trading, unplanned urbanization, environmental and climate change are having a significant impact on disease transmission. Therefore, the Ministry of Health of Nepal had brought some changes in strategies based on activities for disease control, vector control, preventive and preparedness for outbreak response. Consequently, the cases and deaths due to malaria, kala-azar (visceral leishmaniasis), lymphatic filaiasis and dengue fever/dengue hemorrhagic fever have been brought down markedly.

Multidrug resistance Plasmodium falciparum remains a significant global health problem worldwide. New alternative antimalarial drugs are urgently needed. Dioscorea membranacea Pierre. is a Thai-medicinal plant that has been shown to exhibit a wide range of pharmacological activities. The study aimed to investigate antimalarial activity and possible protein targets of action of the crude ethanolic extract of the rhizome of this plant. The in vitro antimalarial activity expressed as IC50 (concentration that inhibits the parasite growth by 50%) of the extract against 3D7 chloroquine-sensitive P. falciparum and K1 chloroquine-resistant P. falciparum clones were 10.1 (8.8-10.3) and 9.3 (9.17-9.63) µg/ml [median (range)], respectively. The cytotoxicity of against the human fibroblast cell OUMS-36T-1F was 96.4 (96.3-96.5) µg/ml. The selectivity index (SI) for the 3D7 and K1 clones was 9.5 and 10.4, respectively. Preliminary investigation of the protein targets of action in 3D7 P. falciparum clone revealed 13 up-regulated protein spots and 14 down-regulated protein spots. For further development of D. membranacea Pierre. as a promising antimalarial drug candidate, identification of these proteins by mass spectrometry and investigation of their mode of antimalarial actions are encouraged. Key words: Malaria, proteomics, Dioscorea membranacea Pierre.

Duffy binding–like domain (DBL) and cysteine-rich interdomain region (CIDR) domain genes of Plasmodium falciparum erythrocyte membrane protein 1 (PfEMP1) encode malaria virulence proteins. The variants of these genes have been reported to be associated with severe/complicated malaria. The present study investigated the prevalence and distribution patterns of DBLα0.6/9, DBLα1.1, DBLα1 not var3 genes, DBLα2/α1.1/2/4/7, DBLβ12 & DBLβ3/5, DBLe8, CIDRα1.4, and CIDRα1.6 of P. falciparum isolates along the Thai-Myanmar border. The association between PfEMP1 variants and parasite density was also investigated. Two hundred and thirteen finger-prick dried blood spot (DBS) or whole blood samples were collected in 2007 and 2015, from patients with acute uncomplicated P. falciparum in Tak, Kanchanaburi, and Ranong provinces. Analysis of the variant genes was performed using polymerase chain reaction (PCR). The DBLs variant which was found at the highest and lowest frequencies in the three provinces were DBLα1 not var3 (72.77%), and DBLe8 (17.37%). The two CIDR domain variants were found at relatively lower frequencies compared with DBL domain variants (9.9% and 30.1%). P. falciparum isolates carrying the four PfEMP1 variants, i.e., DBLα0.6/9, DBLα1.1, DBLα2/α.1.1/2/4/7, and DBLe8 were found to be significantly associated with low parasitemia. Both DBLα0.6/9 and DBLα2/α1.1/2/4/7 variant genes which were present at high frequencies in this border area could be potential candidate markers for predicting P. falciparum hyperparasitemia and in this border area. Furthermore, the information could be exploited as candidate proteins for the development of an effective malaria vaccine in specific malaria-endemic areas.

Objective To investigate cytotoxic activity of ethyl-p-methoxycinnamate (EPMC) including its effect on p-glycoprotein (multidrug resistance-1: mdr-1 gene) in human cholangiocarcinoma cell. Methods Cytotoxic activity of EPMC against human cholangiocarcinoma (CL-6), fibroblast (OUMS-36T-1F), and colon cancer (Caco-2) cell lines were assessed using MTT assay. Selectivity index (SI) was determined as the ratio of IC50 (concentration that inhibits cell growth by 50%) of EPMC in OUMS-36T-1F and that in CL-6 cell. Cell cycle arrest and apoptosis in CL-6 cells were investigated by flow cytometry and fluorescent microscopy. Effect of EPMC on mdr-1 gene expression in CL-6 and Caco-2 was determined by real-time PCR. Results The median (95% CI) IC50 values of EPMC in CL-6, OUMS-36T-1F, and Caco-2 were 245.5 (243.1-266.7), 899.60 (855.8-966.3) and 347.0 (340.3-356.9) µg/ml, respectively. The SI value of the compound for the CL-6 cell was 3.70. EPMC at IC50 inhibited CL-6 cell division and induced apoptosis compared to untreated control. EPMC exposure did not induce mdr-1 gene expression in both CL-6 and Caco-2 cells. Conclusion The results suggest the potential role of EPMC in cholangiocarcinoma with a low possibility of drug resistance induction.

Primaquine is the only antimalarial drug available for eradicating the hypnozoite stage of Plasmodium vivax to prevent the disease from recurring. However, one limitation of its clinical use is the long treatment course of 14 days, which may result in poor patients' adherence and low treatment efficacy. The aim of the current study was to assess patients' adherence and the clinical effectiveness of the unsupervised standard 14-day primaquine regimen (daily dose of 15mg base/kg body weight daily for 14 days) when given together with 3-day chloroquine (25mg base/kg body weight over 3 days). The study was conducted in 85 patients with P. vivax malaria in a malaria endemic area along the Thai-Myanmar border. Patients' adherence to primaquine therapy was assessed based on primaquine concentrations in finger-prick dried blood spot (DBS) samples alongside patients' self-reporting on drug administration and pill counting methods. Results suggest high rate of patients' adherence to this 14-day primaquine regimen (95-98% based on primaquine concentrations in DBS on days 3, 7, and 14 of treatment, and 100% based on patients' self-reporting and pill counting methods. Clinical effectiveness was 100% during the 42-day follow-up.

Malaria remains a public health problem in tropical and subtropical regions. Resistance of Plasmodium falciparum to artemisinins in Southeast Asia is a great concern for disease control and research on discovery and development of new alternative antimalarial drugs is urgently required. In a previous study, the fruit of Piper chaba Hunt. was demonstrated to exhibit promising antimalarial activity against the asexual stage of 3D7 (chloroquine-sensitive) and K1 (chloroquine-resistant) P. falciparum clones. The aim of the present study was to further investigate the antimalarial activity of piperine, the major isolated constituent of Piper chaba Hunt. fruits against both P. falciparum clones. The antimalarial activity was determined using SYBR green-I-based assay and morphological change was observed under the light microscope with Giemsa staining. The median IC50 (concentration that inhibits parasite growth by 50%) values of piperine against 3D7 and K1 P. falciparum were 111.5 and 59 μM, respectively. A marked change in parasite morphology was observed within 48 hours of piperine exposure. Results of real-time PCR showed no effect of piperine on modulating the expression of the three genes associated with antimalarial drug resistance in P. falciparum, i.e., pfcrt, pfmdr1, and pfmrp1. Piperine could be a promising candidate for further development as an antimalarial drug based on its antimalarial potency and low risk of resistance development.

The K13 propeller domain mutation and pfmdr1 amplification have been proposed as useful molecular markers for detection and monitoring of artemisinin resistant Plasmodium falciparum Welch, 1897. Genomic DNA isolates of P. falciparum was extracted from 235 dried blood spot or whole blood samples collected from patients with uncomplicated falciparum malaria residing in areas along the Thai-Myanmar border during 2006-2010. Nested polymerase chain reaction (PCR) and sequencing were performed to detect mutations in K13 propeller domain of P. falciparum at codon 427-709. Pfmdr1 gene copy number was determined by SYBR Green I real-time PCR. High prevalence of pfmdr1 multiple copies was observed (42.5% of isolates). The presence of K13 mutations was low (40/235, 17.2%). Seventeen mutations had previously been reported and six mutations were newly detected. The C580Y was found in two isolates (0.9%). The F446I, N458Y and P574L mutations were commonly detected. Seven isolates had both K13 mutation and pfmdr1 multiple copies. It needs to be confirmed whether parasites harbouring both K13 mutation and pfmdr1 multiple copies and/or the observed new mutations of K13 propeller domain are associated with clinical artemisinin resistance.

Cholangiocarcinoma (CCA) is the cancer of bile duct with high mortality rate particularly in Thailand. The clinical efficacy of the standard chemotherapeutics remains unsatisfactory, and therefore, discovery and development of the new alternative drugs with high efficacy and tolerability is needed. The aim of the study was to investigate cytotoxic activity as well as the underlying mechanisms through which atractylodin and β-eudesmol exert their activities on CCA cell growth inhibition, cell cycle arrest, and cell apoptosis. Effects of the compounds on cell cytotoxicity, cell cycle arrest, and cell apoptosis were analyzed using MTT assay, BD Cycletest™ Plus DNA kit, and FITC Annexin V Apoptosis Detection Kit I, respectively. The cytotoxic activities of both compounds were concentration- and time-dependent. The IC50 [mean (SD)] of atractylodin and β-eudesmol were 41.66 (2.51) and 39.33 (1.15) μg/ml respectively. Both promoted cell cycle arrest at G1 phase, and induced cell apoptosis through activation of caspase-3/7. The highest activity was observed at 48 h of exposure. Results suggest that these mechanisms are at least in part, explain the cell cytotoxic and anti-CCA activity of atractylodin and β-eudesmol shown in vitro and in vivo models. Keywords: Cholangiocarcinoma, Atractylodin, β-Eudesmol, Cytotoxicity, Cell cycle arrest, Cell apoptosis

A 3-day artesunate-mefloquine combination therapy has been using as first-line treatment for acute uncomplicated Plasmodium falciparum malaria in Thailand since 1995 on the background of mefloquine resistance. The aim of the present study was to assess sensitivity of P. falciparum isolates (n=44) in an area along the Thai-Myanmar border (year 2009) to artesunate, mefloquine, chloroquine and quinine, including their correlation with clinico-parasitological response. Twenty, 19, and 5 isolates were collected from patients with 'Adequate Clinical and Parasitological Response (ACPR)', 'Late Parasitological Failure (LPF)' and 're-infection', respectively. The IC50 of artesunate and mefloquine were significantly higher in patients with LPF compared with ACPR and re-infection. The proportion of isolates with declined artesunate or mefloquine sensitivity in the LPF group (47.4%) was significantly higher than the ACPR group (5.0%). A weak but statistical significant correlation (r=0.384, p=0.01) was observed between IC50 values of artesunate and parasite clearance time (PCT). There was no significant relationship between in vitro sensitivity of parasite isolates to chloroquine or quinine and clinical response. In vitro susceptibility of P. falciparum isolates to artesunate and mefloquine may be used as a useful reliable tool to predict clinico-pathological response following a 3-day artesunate-mefloquine combination therapy.

To investigate the distribution and patterns of pfcrt and pfmdr1 polymorphisms in Plasmodium falciparum (P. falciparum) isolates collected from the malaria endemic area of Thailand along Thai-Myanmar border.Dried blood spot samples were collected from 172 falciparum malaria patients prior received treatment. The samples were extracted using chelex to obtain parasite DNA. PCR-RFLP was employed to detect pfcrt mutation at codons 76, 220, 271, 326, 356 and 371, and the pfmdr1 mutation at codon 86. Pfmdr1 gene copy number was determined by SYBR Green I real-time PCR.Mutant alleles of pfcrt and wild type allele of pfmdr1 were found in almost all samples. Pfmdr1 gene copy number in isolates collected from all areas ranged from 1.0 to 5.0 copies and proportion of isolates carrying1 gene copies was 38.1%. The distribution and patterns of pfcrt and pfmdr1 mutations were similar in P. falciparum isolates from all areas. However, significant differences in both number of pfmdr1 copies and prevalence of isolates carrying1 gene copies were observed among isolates collected from different areas. The median pfmdr1 copy number in P. falciparum collected from Kanchanaburi and Mae Hongson were 2.5 and 2.0, respectively and more than half of the isolates carried1 gene copies.The observation of pfmdr1 wild type and increasing of gene copy number may suggest declining of artesunate-mefloquine treatment efficacy in P. falciparum isolates in this border area.

Background A markedly high failure rate of three-day artesunate-mefloquine was observed in the area along the Thai-Myanmar border. Methods Identification of Plasmodium falciparum isolates with intrinsic resistance to each component of the artesunate-mefloquine combination was analysed with integrated information on clinico-parasitological response, together with systemic drug exposure (area under blood/plasma concentration-time curves (AUC)) of dihydroartemisinin and mefloquine, and in vitro sensitivity of P. falciparum in a total of 17 out of 29 P. falciparum isolates from patients with acute uncomplicated falciparum malaria. Analysis of the contribution of in vitro parasite sensitivity and systemic drug exposure and relationship with pfmdr1 copy number in the group with sensitive response was performed in 21 of 69 cases. Results Identification of resistance and/or reduced intrinsic parasitocidal activity of artesunate and/or mefloquine without pharmacokinetic or other host-related factors were confirmed in six cases: one with reduced sensitivity to artesunate alone, two with resistance to mefloquine alone, and three with reduced sensitivity to artesunate combined with resistance to mefloquine. Resistance and/or reduced intrinsic parasitocidal activity of mefloquine/artesunate, together with contribution of pharmacokinetic factor of mefloquine and/or artesunate were identified in seven cases: two with resistance to mefloquine alone, and five with resistance to mefloquine combined with reduced sensitivity to artesunate. Pharmacokinetic factor alone contributed to recrudescence in three cases, all of which had inadequate whole blood mefloquine levels (AUC0-7days). Other host-related factors contributed to recrudescence in one case. Amplification of pfmdr1 (increasing of pfmdr1 copy number) is a related molecular marker of artesunate-mefloquine resistance and seems to be a suitable molecular marker to predict occurrence of recrudescence. Conclusions Despite the evidence of a low level of a decline in sensitivity of P. falciparum isolates to artemisinins in areas along the Thai-Myanmar border, artemisinin-based combination therapy (ACT) would be expected to remain the key anti-malarial drug for treatment of multidrug resistance P. falciparum. Continued monitoring and active surveillance of clinical efficacy of ACT, including identification of true artemisinin resistant parasites, is required for appropriate implementation of malaria control policy in this area.