In vitro sensitivity of Plasmodium falciparum from China-Myanmar border area to major ACT drugs and polymorphisms in potential target genes

Drug resistance has always been one of the most important impediments to global malaria control. Artemisinin resistance has recently been confirmed in the Greater Mekong Subregion (GMS) and efforts for surveillance and containment are intensified. To determine potential mechanisms of artemisinin resistance and monitor the emergence and spread of resistance in other regions of the GMS, we investigated the in vitro sensitivity of 51 culture-adapted parasite isolates from the China-Myanmar border area to four drugs. The 50% inhibitory concentrations (IC₅₀s) of dihydroartemisinin, mefloquine and lumefantrine were clustered in a relatively narrow, 3- to 6-fold range, whereas the IC₅₀ range of artesunate was 12-fold. We assessed the polymorphisms of candidate resistance genes pfcrt, pfmdr1, pfATP6, pfmdr6 and pfMT (a putative metabolite/drug transporter). The K76T mutation in pfcrt reached fixation in the study parasite population, whereas point mutations in pfmdr1 and pfATP6 had low levels of prevalence. In addition, pfmdr1 gene amplification was not detected. None of the mutations in pfmdr1 and pfATP6 was associated significantly with in vitro sensitivity to artemisinin derivatives. The ABC transporter gene pfmdr6 harbored two point mutations, two indels, and number variations in three simple repeats. Only the length variation in a microsatellite repeat appeared associated with altered sensitivity to dihydroartemisinin. The PfMT gene had two point mutations and one codon deletion; the I30N and N496– both reached high levels of prevalence. However, none of the SNPs or haplotypes in PfMT were correlated significantly with resistance to the four tested drugs. Compared with other parasite populations from the GMS, our studies revealed drastically different genotype and drug sensitivity profiles in parasites from the China-Myanmar border area, where artemisinins have been deployed extensively for over 30 years.     

Plasmodium falciparum: in vitro interactions of artemisinin with amodiaquine, pyronaridine, and chloroquine

In the scenario of drug-resistant Plasmodium falciparum malaria combination therapy represents an effective approach. Artemisinin and its derivatives are of special interest because they represent the most effective group of compounds against multidrug-resistant malaria with a rapid onset of action and a short half-life. Interactions of artemisinin with amodiaquine, pyronaridine, and chloroquine were therefore investigated against three strains of P. falciparum using a 48-h in vitro culture assay. Two of the strains were chloroquine sensitive and one was partially chloroquine resistant. Observed effective concentrations (O) of the combined compounds at different concentration ratios were calculated for different degrees of inhibition (EC50, EC90, EC99) and compared to expected calculated effective concentrations (E) using a probit method. Synergism with mean O/E EC90 values of 0.25 and 0.8 were found with the combination of artemisinin and the two Mannich bases, amodiaquine and pyronaridine, respectively, whereas chloroquine showed addition with a mean value of 1.2. Although both amodiaquine and chloroquine are 4-aminoquinolines, their interaction with artemisinin appears to be different. The combination of artemisinin with amodiaquine represents an important option for the treatment of falciparum malaria.     

Susceptibility of Colombian Plasmodium falciparum isolates to 4-aminoquinolines and the definition of amodiaquine resistance in vitro

There are wide variations in the threshold used to define in vitro resistance of Plasmodium falciparum to amodiaquine (AQ), probably due to differences in methodology and interpretation. In vitro susceptibility data of Colombian P. falciparum strains to AQ and N-desethylamodiaquine is used to illustrate the need to standardized methodologies and compare inhibitory concentrations, instead of resistant/susceptible phenotypes, when studying the mechanisms of resistance to AQ and monitoring drug susceptibility trends in the field.     

Crystal structure of phosphoethanolamine methyltransferase from Plasmodium falciparum in complex with amodiaquine

Phosphoethanolamine N-methyltransferase (PMT) is essential for phospholipid biogenesis in the malarial parasite Plasmodium falciparum. PfPMT catalyzes the triple methylation of phosphoethanolamine to produce phosphocholine, which is then used for phosphatidylcholine synthesis. Here we describe the 2.0? resolution X-ray crystal structure of PfPMT in complex with amodiaquine. To better characterize inhibition of PfPMT by amodiaquine, we determined the IC(50) values of a series of aminoquinolines using a direct radiochemical assay. Both structural and functional analyses provide a possible approach for the development of new small molecule inhibitors of PfPMT.     

Stage-specific sensitivity of Plasmodium falciparum to antifolates

Highly synchronous cultures of Plasmodium falciparum were exposed to therapeutic concentrations of sulfadoxine or pyrimethamine at different developmental stages to investigate the effect on subsequent growth. Morphological observations showed that schizont formation from uninuclear trophozoites was the only process inhibited by the drugs. Segmentation of mature schizonts, merozoite invasion and development of the ring stage remained unaffected. These results support earlier reports suggesting that DNA synthesis is most pronounced in 32-42 h old trophozoites. The possible relevance of our results to the metabolism of P. falciparum is discussed.     

Polymorphism in the Plasmodium falciparum chloroquine-resistance transporter protein links verapamil enhancement of chloroquine sensitivity with the clinical efficacy of amodiaquine

Background

Chloroquine accumulates in the acidic digestive vacuole of the intraerythrocytic malaria parasite, and prevents the detoxication of haematin released during haemoglobin digestion. Changes in protein PfCRT in the digestive vacuole membrane of growing intra-erythrocytic stages of Plasmodium falciparum are crucial for resistance. Expressed in yeast, PfCRT resembles an anion channel. Depressed anion channel function could increase intralysosomal pH to reduce entry of basic drug, or enhanced function could reduce drug interaction with target haematin. The most important resistance-associated change is from positively-charged lysine-76 to neutral threonine which could facilitate drug efflux through a putative channel. It has been proposed that the resistance-reversing effect of verapamil is due to hydrophobic binding to the mutated PfCRT protein, and replacement of the lost positive charge, which repels the access of 4-aminoquinoline cations, thus partially restoring sensitivity. Desethylamodiaquine, the active metabolite of amodiaquine, which has significant activity in chloroquine-resistance, may also act similarly on its own.

Methods

Changes in physicochemical parameters in different CQ-resistant PfCRT sequences are analysed, and correlations with drug activity on lines transfected with different alleles of the pfcrt gene are examined.

Results and conclusions

The results support the idea that PfCRT is a channel which, in resistant parasites, can allow efflux of chloroquine from the digestive vacuole. Activity of the chloroquine/verapamil combination and of desethylamodiaquine both correlate with the mean hydrophobicity of PfCRT residues 72-76. This may partly explain clinical-resistance to amodiaquine found in the first chloroquine-resistant malaria cases from South America and enables tentative prediction of amodiaquine's clinical activity against novel haplotypes of PfCRT.     

In vitro activity of immunosuppressive drugs against Plasmodium falciparum

Strong evidence suggests that quality strategic behaviour change communication (BCC) can improve malaria prevention and treatment behaviours. As progress is made towards malaria elimination, BCC becomes an even more important tool. BCC can be used 1) to reach populations who remain at risk as transmission dynamics change (e.g. mobile populations), 2) to facilitate identification of people with asymptomatic infections and their compliance with treatment, 3) to inform communities of the optimal timing of malaria control interventions, and 4) to explain changing diagnostic concerns (e.g. increasing false negatives as parasite density and multiplicity of infections fall) and treatment guidelines. The purpose of this commentary is to highlight the benefits and value for money that BCC brings to all aspects of malaria control, and to discuss areas of operations research needed as transmission dynamics change.     

Blood schizontocidal activity of methylene blue in combination with antimalarials against Plasmodium falciparum

Methylene blue (MB) is the oldest synthetic antimalarial. It is not used anymore as antimalarial but should be reconsidered. For this purpose we have measured its impact on both chloroquine sensitive and resistant Plasmodium strains. We showed that around 5 nM of MB were able to inhibit 50% of the parasite growth in vitro and that late rings and early trophozoites were the most sensitive stages; while early rings, late trophozoites and schizonts were less sensitive. Drug interaction study following fractional inhibitory concentrations (FIC) method showed antagonism with amodiaquine, atovaquone, doxycycline, pyrimethamine; additivity with artemether, chloroquine, mefloquine, primaquine and synergy with quinine. These results confirmed the interest of MB that could be integrated in a new low cost antimalarial combination therapy.     

Increased sensitivity to the antimalarials mefloquine and artemisinin is conferred by mutations in the pfmdr1 gene of Plasmodium falciparum

The declining efficacy of chloroquine and pyrimethamine/sulphadoxine in the treatment of human malaria has led to the use of newer antimalarials such as mefloquine and artemisinin. Sequence polymorphisms in the pfmdr1 gene, the gene encoding the plasmodial homologue of mammalian multidrug resistance transporters, have previously been linked to resistance to chloroquine in some, but not all, studies. In this study, we have used a genetic cross between the strains HB3 and 3D7 to study inheritance of sensitivity to the structurally unrelated drugs mefloquine and artemisinin, and to several other antimalarials. We find a complete allelic association between the HB3-like pfmdr1 allele and increased sensitivity to these drugs in the progeny. Different pfmdr1 sequence polymorphisms in other unrelated lines were also associated with increased sensitivity to these drugs. Our results indicate that the pfmdr1 gene is an important determinant of susceptibility to antimalarials, which has major implications for the future development of resistance.     

Over-production of lactate dehydrogenase from Plasmodium falciparum opens a route to new antimalarials

Over-production of lactate dehydrogenase (PfLDH) from Plasmodium falciparum from E. coli TG2 cells transformed with a pKK223-3 plasmid containing the wild type gene isolated by Bzik DJ, Fox BA, and Gonyer K (1993) Mol. Biochem. Parasit. 59, 155–166, gave mostly an inactive protein after isolation. Sequencing the N-terminus of the over-produced protein showed that the major product commenced at an internal methionine. Truncation of the protein occurred due to the inappropriate priming from a Shine–Dalgarno (SD) sequence upstream of Met 35. Silent mutations of this SD sequence to remove the purine-rich region allowed over-production of the full length PfLDH up to 15 mg protein l^–1 broth. The purified protein exhibited biochemical properties of an authentic LDH enzyme. However, high activity with 3-acetylpyridine adenine dinucleotide as well as with the natural cofactor, NAD, was also observed. The high-resolution X-ray structure obtained from the recombinant enzyme has provided the opportunity for the development of inhibitors specific to PfLDH.     

In vitro efficiency of new acridyl derivatives against Plasmodium falciparum

A series of new 9-substituted acridyl derivatives were synthesized and their in vitro antimalarial activity was evaluated against one chloroquine-sensitive strain (3D7) and three chloroquine-resistant strains [W2 (Indochina), Bre1 (Brazil) and FCR3 (Gambia)] of Plasmodium falciparum. Some compounds inhibit the growth of malarial parasite with IC50 相似文献   

In vitro responses of human peripheral blood mononuclear cells to Plasmodium falciparum antigen

Immunity to malaria involves cell-mediated and humoral responses. The cell-mediated reaction is thought to focus particularly on the activity of cells of the macrophage lineage. The ability of antigen-stimulated human peripheral blood lymphocytes to undergo proliferation and produce factors capable of causing macrophage inhibition of parasite growth has been examined. While lymphocyte proliferation, gamma-interferon production, and anti-malarial antibody levels of malaria-exposed Papua New Guinea donors were correlated, and significantly different from Australian subjects, macrophage parasite inhibition was no different in these two groups. Further, there was no evidence for acquired, persistent, cell-mediated immunity, as judged by the monocyte procoagulant test. The results are discussed within the context of human acquired resistance to malaria.     

Longitudinal in vitro surveillance of Plasmodium falciparum sensitivity to common anti-malarials in Thailand between 1994 and 2010

ABSTRACT: BACKGROUND: Drug and multidrug-resistant Plasmodium falciparum malaria has existed in Thailand for several decades. Furthermore, Thailand serves as a sentinel for drug-resistant malaria within the Greater Mekong sub-region. However, the drug resistance situation is highly dynamic, changing quickly over time. Here parasite in vitro drug sensitivity is reported for artemisinin derivatives, mefloquine, chloroquine and quinine, across Thailand. METHODS: Blood was drawn from patients infected with P. falciparum in seven sentinel provinces along Thai international borders with Cambodia, Myanmar, Laos, and Malaysia. In vitro parasite sensitivity was tested using the World Health Organization's microtest (mark III) (between 1994 and 2002) and the histidine-rich protein-2 (HRP2)-based enzyme-linked immunosorbent assay (in 2010). Following World Health Organization protocol, at least 30 isolates were collected for each province and year represented in this study. Where possible, t-tests were used to test for significant differences. RESULTS: There appears to be little variation across study sites with regard to parasite sensitivity to chloroquine. Quinine resistance appears to have been rising prior to 1997, but has subsequently decreased. Mefloquine sensitivity appears high across the provinces, especially along the north-western border with Myanmar and the eastern border with Cambodia. Finally, the data suggest that parasite sensitivity to artemisinin and its derivatives is significantly higher in provinces along the north-western border with Myanmar. CONCLUSIONS: Parasite sensitivity to anti-malarials in Thailand is highly variable over time and largely mirrors official drug use policy. The findings with regard to reduced sensitivity to artemisinin derivatives are supported by recent reports of reduced parasite clearance associated with artemisinin. This trend is alarming since artemisinin is considered the last defence against malaria. Continued surveillance in Thailand, along with increased collaboration and surveillance across the entire Greater Mekong sub-region, is clearly warranted.     

In vitro beta-hematin formation assays with plasma of mice infected with Plasmodium yoelii and other parasite preparations: comparative inhibition with quinoline and endoperoxide antimalarials.

Formation of beta-hematin in vitro could be catalyzed in the presence of various preparations related to the malaria parasite viz., the cell free homogenate of Plasmodium yoelii, lipid extract of the parasite homogenate, purified malarial hemozoin and synthetic beta-hematin. Plasma from mice infected with P. yoelii also catalyzed in vitro beta-hematin formation with highly significant efficiency. The plasma based beta-hematin formation assay was highly sensitive, as the background absorbance was almost negligible due to absence of any preformed hemozoin. The plasma beta-hematin synthesizing activity was recovered in the lipid extract. The quinoline and endoperoxide antimalarials act by inhibiting hemozoin biosynthesis in the malaria parasite. Therefore, the in vitro beta-hematin formation assay is useful for the screening and identification of blood schizontocidal antimalarials acting through interruption of heme detoxification in the parasite. Quinoline and endoperoxide antimalarials showed about three fold greater inhibition of beta-hematin synthesizing activity in the plasma-based assays as compared to that of P. yoelii homogenate-based assays. The specificity of the inhibition was similar in both preparations. The plasma-based assay therefore provides a better alternative than the parasite homogenate-based assay for in vitro screening and identification of novel inhibitors of hemozoin biosynthesis as potential blood schizontocidal antimalarials.     

Premature release of Plasmodium falciparum from swollen erythrocytes induced by some antimalarials

Qinghaosu and chloroquine, but not pyrimethamine, treatment of Plasmodium falciparum cultures resulted in the formation of swollen red blood cells (RBCs) and the expulsion of degenerate trophozoites and schizonts, but not ring-stage parasites, from the infected RBCs. The parasite release resulted in the formation of RBCs with holes, that had otherwise retained their structural integrity. Membranes of swollen RBCs and their ghosts associated with parasites were efficiently visualized by Giemsa staining of thin smears for 18-24 hr but not by standard Giemsa staining for 20 min.     

Mechanisms of in vitro resistance to dihydroartemisinin in Plasmodium falciparum

The recent reports of artemisinin (ART) resistance in the Thai-Cambodian border area raise a serious concern on the long-term efficacy of ARTs. To elucidate the resistance mechanisms, we performed in vitro selection with dihydroartemisinin (DHA) and obtained two parasite clones from Dd2 with more than 25-fold decrease in susceptibility to DHA. The DHA-resistant clones were more tolerant of stressful growth conditions and more resistant to several commonly used antimalarial drugs than Dd2. The result is worrisome as many of the drugs are currently used as ART partners in malaria control. This study showed that the DHA resistance is not limited to ring stage, but also occurred in trophozoites and schizonts. Microarray and biochemical analyses revealed pfmdr1 amplification, elevation of the antioxidant defence network, and increased expression of many chaperones in the DHA-resistant parasites. Without drug pressure, the DHA-resistant parasites reverted to sensitivity in approximately 8 weeks, accompanied by de-amplification of pfmdr1 and reduced antioxidant activities. The parallel decrease and increase in pfmdr1 copy number and antioxidant activity and the up and down of DHA sensitivity strongly suggest that pfmdr1 and antioxidant defence play a role in in vitro resistance to DHA, providing potential molecular markers for ART resistance.     

Plasmodium falciparum: in vitro induction of resistance to aminopterin

Plasmodium falciparum parasites were grown on microplates in the presence of aminopterin. The FCR-8 strain was more sensitive to aminopterin than a Richards strain and died within 1 week of treatment. A few parasites of the Richards strain survived treatment and developed normal parasitemias. This strain was resistant to aminopterin at concentrations not higher than those used for its selection. Removal of aminopterin did not affect the growth of the resistant variant, showing that it was not aminopterin dependent. Aminopterin affected the sensitive parasites by interfering with nucleic acid synthesis, whereas protein synthesis was not impaired. Gametocytogenesis was unaffected by aminopterin.