Optimized in vitro production of Plasmodium vivax ookinetes

Previous reports have described obtaining mature Plasmodium vivax ookinetes in vitro using blood from infected patients using a simplified, field-based protocol. Here, we report protocols that produce improved P. vivax ookinete yields and morphological development. Optimal conditions included induction of gametogenesis using 10 mM Tris, 170 mM NaCl, 10 mM glucose, 25 mM NaHCO(3), and 100 μM xanthurenic acid for 90 minutes at pH 8.0-8.2, followed by culture in RPMI-1640, 50 mg/mL hypoxanthine, 25 mM HEPES, 29 mM NaHCO(3), 2 mM L-glutamine, and 20% fetal bovine serum at pH 8.4 for 36 hours. Ookinetes were produced in 86% (18/21) of optimized in vitro cultures; yields ranged from 6.5 × 10(4) to 2.8 × 10(6); percent gametocyte conversion ranged from 1.4% to 4.7%. This improved method is suitable for preparation of P. vivax ookinetes in quantities sufficient for biochemical, molecular, and cell biological analysis where basic laboratory facilities are in proximity to patients with vivax malaria.     

Plasmodium falciparum ookinetes require mosquito midgut chondroitin sulfate proteoglycans for cell invasion

Malaria transmission entails development of the Plasmodium parasite in its insect vector, the Anopheles mosquito. Parasite invasion of the mosquito midgut is the critical first step and involves adhesion to host epithelial cell ligands. Partial evidence suggests that midgut oligosaccharides are important ligands for parasite adhesion; however, the identity of these glycans remains unknown. We have identified a population of chondroitin glycosaminoglycans along the apical midgut microvilli of Anopheles gambiae and further demonstrated ookinete recognition of these glycans in vitro. By repressing the expression of the peptide-O-xylosyltransferase homolog of An. gambiae by means of RNA interference, we blocked glycosaminoglycan chain biosynthesis, diminished chondroitin sulfate levels in the adult midgut, and substantially inhibited parasite development. We provide evidence for the in vivo role of chondroitin sulfate proteoglycans in Plasmodium falciparum invasion of the midgut and insight into the molecular mechanisms mediating parasite-mosquito interactions.     

In vitro activity of quinolines against Plasmodium falciparum in Gabon

The assessment of drug sensitivity of Plasmodium falciparum to antimalarial drugs is of vital interest for malaria endemic regions. We conducted a follow-up study to monitor the in vitro activity of the most commonly used quinolines against fresh P. falciparum isolates in Lambaréné, Gabon by measuring schizont maturation inhibition in 2002. Mean 50% effective concentration levels for chloroquine, quinine, and mefloquine were 5.5micromol/l blood, 286nmol/l blood medium mixture (BMM), and 1.1micromol/l blood, respectively. All isolates (n=40) were found to be highly resistant to chloroquine. One isolate was resistant to mefloquine and five isolates were presenting borderline-resistance. All isolates were inhibited by quinine concentrations below the threshold of resistance (n=43).Besides the observation of an increasing number of borderline resistant isolates to mefloquine, an extremly high parasite resistance to chloroquine-still officially the first line antimalarial in Gabon-seems to be of particular concern.     

In vitro sensitivity of Plasmodium falciparum to sulfadoxine and pyrimethamine

An in vitro microtechnique of Rieckmann et al., (1978) modified by Yisunsri and Rieckmann (1980) using 3 media; Waymouth, Waymouth plus 10% human serum, and RPMI was assessed to determine the sensitivity of P. falciparum to sulfadoxine, pyrimethamine and its combination. The study confirmed the synergism between sulfadoxine and pyrimethamine. There was no interaction between media and drug tested. MIC1 and MIC2 of sulfadoxine in different media showed significant difference (p less than 0.001). No significant difference was observed in MIC1 and MIC2 of pyrimethamine in the three media used (p greater than 0.05). For sulfadoxine-pyrimethamine combination, MIC1 and MIC2 in Waymouth alone and plus 10% human serum showed no significance (p greater than 0.05) while in RPMI showed positive correlation (p less than 0.001). MIC1 might be more applicable for clinical evaluation than MIC2. At present Waymouth medium with 5% patient serum, is considered to be the most suitable for testing sensitivity of malarial parasites.     

In vitro chemosensitivity of Plasmodium falciparum to four chloroquine derivatives

The antimalarial activity of four chloroquine derivatives has been assessed in vitro by the Trager and Jensen technique against the strain of Plasmodium falciparum FCC, 2spp. Monodesethyl-chloroquine possessed a significant activity, reducing the parasitaemia to 5% with 2 nM ml-1 (base). The hydroxy-metabolite showed a slight activity, reducing the parasitaemia to 39.5% with 2 nM ml-1 (base). No activity was found with the amino-metabolite and the pyrrolidinyl chemical derivative. The anti-malarial activity of monodesethyl-chloroquine should be considered for pharmacokinetics and for optimizing chloroquine treatments.     

云南省恶性疟原虫抗萘酚喹株的体外培育

目的培育恶性疟原虫萘酚喹抗性虫株,为恶性疟原虫抗性的深入研究提供实验虫株。方法采用恶性疟原虫FccSM/YN株,用Trager法进行体外连续培养,待其正常生长后,在培养基中间断添加不同浓度的萘酚喹进行克隆筛选,培育抗性,在培育前及用药后不同时段同步化处理疟原虫,使疟原虫小环状体率达95%以上,然后用Rieckmann体外微量法测定萘酚喹对培养虫株的半数抑制浓度(IC50)。结果药物刺激前(亲代)IC50为3.03 nmol/L;药物刺激后166 d IC50为43.07 nmol/L,为药物刺激前的14.22倍;停止药物刺激后25 d的IC50为18.98 nmol/L,较药物刺激后166 d下降55.94%。但仍然较亲代高6.26倍。结论连续体外培养药物间断刺激方法可以筛选培育出高度抗萘酚喹恶性疟原虫虫株。该株恶性疟原虫抗性不稳定,停药后抗性程度有所下降。     

萘酚喹单用及其与阿奇霉素伍用体外抑制恶性疟原虫作用研究

目的比较萘酚喹单用及其与阿奇霉素伍用体外对恶性疟原虫的作用。方法采用Riekmann体外微量法测定恶性疟原虫对上述药物的敏感性,将单一药物组的半数抑制量(ID50)与伍用药物组进行对比研究。结果萘酚喹、阿奇霉素单用对恶性疟原虫萘酚喹敏感株的ID50分别为2.11和3.21 nmol/L,萘酚喹与阿奇霉素伍用对恶性疟原虫萘酚喹敏感株的ID50为0.18和0.15 nmol/L,协同效应(FIC)值为0.14;单用对恶性疟原虫萘酚喹抗性株的ID50为57.62和55.21 nmol/L;萘酚喹、阿奇霉素伍用对恶性疟原虫萘酚喹抗性株的ID50为7.07和6.06 nmol/L,FIC值为0.28。配伍药物中各药的FIC值总和均小于1。结论萘酚喹、阿奇霉素伍用对杀灭恶性疟原虫萘酚喹敏感株和抗性株有增效作用。     

In vitro activity of pyronaridine against African strains of Plasmodium falciparum.

The in vitro activity of pyronaridine was determined and compared with the activity of monodesethylamodiaquine and amopyroquine against 31 clinical isolates and two clones of Plasmodium falciparum originating from Central and West Africa using a semi-micro drug susceptibility test. Pyronaridine and amopyroquine were 2.5 and four times less active, respectively, against the highly chloroquine-resistant clone, than against the chloroquine-susceptible clone but were equally active against chloroquine-susceptible and chloroquine-resistant clinical isolates. Compared with chloroquine-susceptible isolates, monodesethylamodiaquine was three times less active against chloroquine-resistant parasites. Pyronaridine is highly active against chloroquine-resistant strains of P. falciparum and may be a promising candidate for the treatment of resistant malaria.     

In vitro inhibition of intracellular growth of Plasmodium falciparum by immune sera

Beginning at the ring stage, synchronized cultures of Plasmodium falciparum were grown in suspension for 22-32 hours. Intracellular growth was assayed by measuring cellular uptake and incorporation into protein of 35S-methionine. Low concentrations (2%) of serum from immune humans and Aotus monkeys were found to inhibit the uptake of the 35S-methionine. The amount of inhibition for a given serum was often inversely related to its indirect fluorescent antibody test titer. Inhibition occurred during the trophozoite stage and was not obtained with a clone lacking the erythrocyte modifications referred to as knobs. Thus, a sensitive new assay is described which allows detection of factors in immune primate sera which can affect maturation of P. falciparum within the erythrocyte. These serum factors are likely to be antibodies which react with antigens expressed at the trophozoite stage on the surface of K+-infected erythrocytes.     

In vitro reversal of chloroquine resistance in Plasmodium falciparum with dihydroethanoanthracene derivatives

The effects of combining four dihydroethanoanthracenic (DEA) derivatives and chloroquine were assessed in vitro against Plasmodium falciparum chloroquine resistant parasites W2, Palo Alto, FCR3, and Bres1. Like verapamil or promethazine, the four dihydroethanoanthracenic derivatives tested can be added to the growing list of agents that show capability in enhancing the activity of chloroquine against resistant parasites. The structurally related tricyclic antihistaminic compounds examined in this study exerted different intrinsic antimalarial activity, but the same chloroquine-potentiating activity as verapamil or promethazine. They may act both on the rate of chloroquine accumulation and on its access to ferriprotoporphyrin IX. The reversal mechanism would be assumed to result from competition between DEA derivatives and chloroquine for efflux translocation sites, thus causing an increase in steady-state accumulation of chloroquine and a return to susceptibility. Restoration of therapeutic efficacy of chloroquine against resistant parasites by the administration of an additional drug available at relatively low cost may be a more effective strategy than the introduction of another antimalarial drug at the national level.     

体外微量法测定恶性疟原虫对抗疟药敏感性的影响因素

目的探讨体外微量法测定恶性疟原虫对抗疟药敏感性的影响因素。方法采用现场测试用的培养基和抗疟药涂药板为材料,用实验室连续培养多年的恶性疟原虫FCC-1/HN株及恶性疟现症病人含虫血进行测定,观察其各种影响因素。结果4℃保存,安瓿封装液体培养基和冰冻干燥培养基分别在2个月和1年内效果不变,超过上述时间,培养基支持疟原虫生长发育的能力将下降。氯喹板2年内、哌喹板6个月内效果稳定,咯萘啶板和青蒿琥酯板保存期超过3个月,效果将会变化。密封涂药板的胶带纸只可1次启封使用,否则会影响测定结果。制作涂药板的塑料应选择对疟原虫生长发育无影响的原材料。4℃保存的药液超过2wk其浓度会发生变化。用于测定的疟原虫应为同步环状体阶段疟原虫,密度以1000～80000个/μl血为宜,含虫血室温保存不超过1h,4℃保存不超过48h。操作技术需熟练,应严格按照操作规范进行,否则会影响测定结果的准确性。结论涂药板、培养基、密封胶带纸、疟原虫及操作技术等均可影响体外微量法测定结果。为体外微量法所用材料及操作技术的标准化和规范化提供参考。     

In vitro activity of artemisone compared with artesunate against Plasmodium falciparum.

Artemisinins show the potential for neurotoxicity in preclinical studies. Artemisone is a leading candidate of second-generation semi-synthetic artemisinin derivatives for antimalarial therapy devoid of neurotoxicity. Artemisone showed 3-5-fold higher in vitro activity (50% effective concentration (EC50) = 0.14 nmol/L, EC90 = 2.55 nmol/L) than artesunate against fresh Plasmodium falciparum isolates from Gabon and a high-activity correlation indicates a shared drug target.     

In vitro damage of cultured ookinetes of Plasmodium gallinaceum by digestive proteinases from susceptible Aedes aegypti.

After exposure to extracts from blood fed A. aegupti cultured ookinetes of P. gallinaceum were damaged to various, defined extents. Immature ookinetes were found to be more sensitive to damage than mature ones. The damage was dependent on the digestion time after which the Aedes extracts had been prepared and could be correlated with the proteolytic activity in the extracts. Control experiments demonstrated that the factors responsible for damage were neither present in unfed mosquitoes nor in blood alone and that the damage was not a result of osmotic stress. After the treatment of the Aedes extracts with lima bean trypsin inhibitor the ookinete damage was much less, indicating that the Aedes trypsin was the major agent of damage. These results were supported by experiments in which the tryptic activity of the extracts was eliminated by thermal denaturation. It is concluded that in the mosquito midgut most of the ookinetes are damaged by digestive enzymes and that this is one factor leading to the discrepancy between the number of ingested macrogametocytes and the number of oocysts which is usually found in nature. It seems that the only ookinetes which have a chance of surviving are those which develop in the centre of the blood clot, away from the site of enzyme action.     

In vitro antiplasmodial activity of Clathria vulpina sponge associated bacteria against Plasmodium falciparum

ObjectiveTo identify the possible antiplasmodial drugs from bacteria associated with marine sponge Clathria vulpina (C. vulpina).MethodsThe C. vulpina samples were collected from Thondi coast and subjected to enumeration and isolation of associated bacteria. Filtered and sterilized extracts (100, 50, 25, 12.5, 6.25 and 3.125 μg/mL) from isolated bacterial strains were screened for antiplasmodial activity against Plasmodium falciparum. Potential extracts were also screened for biochemical constituents.ResultsThirty one bacterial strains were isolated from twelve sponge samples collected from Thondi coast and screened for antiplasmodial assay. The count of bacterial strains were maximum in November 2007 (19×10⁴ CFU/g) and the average count was maximum during the monsoon season (110×10³ CFU/g). The antiplasmodial activity of strain THB15 was highly comparable (IC₅₀ = 20.73 μg/mL) with the positive control chloroquine (IC₅₀ = 19.59 μg/mL) and 21 bacterial strains showed IC₅₀ value of more than 100 μg/mL. Statistical analysis reveals that, significant in vitro antiplasmodial activity (P<0.05) was observed between the concentrations and time of exposure. The chemical injury to erythrocytes showed no morphological changes in erythrocytes by the ethyl acetate extract of bacterial strains after 48 h of incubation. The in vitro antiplasmodial activity might be due to the presence of sugars and alkaloids in the ethyl acetate extracts of bacterial strains.ConclusionThe ethyl acetate extract of THB15 possesses lead compounds for the development of antiplasmodial drugs.     

恶性疟原虫氯喹敏感株与抗性株对青蒿素类药物体外敏感性的研究

目的 测定恶性疟原虫氯喹敏感株与抗性株对青蒿素类药物的体外敏感性. 方法 运用体外微量法与酶联免疫吸附试验(enzyme-linked immunosorbent assay,ELISA)测定青蒿琥酯、蒿甲醚及双氢青蒿素等3种青蒿素类抗疟药物对体外培养的恶性疟原虫氯喹敏感株与氯喹抗性株的体外敏感性,并比较两种方法测定的IC50值. 结果 体外微量法测定的3种药物对恶性疟原虫氯喹敏感株的IC50值依次为3.12 nmol/L、4.30 nmol/L、2.18 nmol/L,对恶性疟原虫氯喹抗性株的IC50值依次为4.31nmol/L、3.90 nmol/L、3.17 nmol/L;同时,将体外微量法与ELISA法所获的结果进行相关性分析,两种方法结果基本一致(r2=0.93,P＜0.001). 结论 恶性疟原虫氯喹抗性株对青蒿素类药物无明显的交叉抗性;ELISA法可用于恶性疟原虫对抗疟药物的体外敏感性检测.     

酮替芬和赛庚啶增强体外培养的恶性疟原虫氯喹抗性株对氯喹反应性的研究

目的 研究酮替芬和赛庚啶增强体外培养的恶性疟原虫氯喹抗性株对氯喹反应性，及其增效机制。 方法 恶性疟原虫氯喹抗性株(Fcc SM1/yN株)取自云南省思茅地区恶性疟患者静脉血，经同步化处理，用新鲜血将红细胞感染率调至0.5%～1.0%，红细胞压积与培养基体积比(红细胞比容)为1 ∶ 9，混匀。制备氯喹药板及氯喹/酮替芬（或氯喹/赛庚啶）组合药板。氯喹药板自上而下每行氯喹终浓度依次为0.312 5～2 560 nmol/L，呈2倍递增。氯喹/酮替芬（或氯喹/赛庚啶）组合药板，是在氯喹药板基础上加入酮替芬（或赛庚啶），每行10孔自左至右终浓度依次为9.77～5 000 nmol/L，呈2倍递增, 每块药板均设A行空白对照。每孔加混匀血样 50 μl，37 ℃ 培养 34 h，镜检计数每200个疟原虫中含3个核以上裂殖体数，计算氯喹单药及各配伍组对恶性疟原虫的半数抑制浓度（IC₅₀），以及酮替芬（或赛庚啶）提高氯喹活性的指数（AEI）。选择AEI值较高的配伍组，进行增效时序性研究。当氯喹对虫体作用 0～10 h 分别加入酮替芬（或赛庚啶），34 h后检测和计算各时间段IC₅₀及AEI。选择氯喹/酮替芬（或氯喹/赛庚啶）最佳配伍剂量，培养恶性疟原虫 20 h，提取总RNA，用实时荧光定量PCR(real-time PCR)分析药物作用前后恶性疟原虫氯喹抗性转移基因（pfcrt）和多药抗性基因（pfmdr1）表达水平。 结果 0.312 5～2 560 nmol/L氯喹与625 nmol/L 酮替芬（或赛庚啶）配伍，增效作用显著，氯喹/酮替芬的IC50为74.53 nmol/L，AEI为0.42；氯喹/赛庚啶的IC50为89.70 nmol/L、AEI为0.30。5 nmol/L氯喹作用 6～7 h 加入625 nmol/L酮替芬（或赛庚啶），增效作用显著，氯喹/酮替芬的IC50为 67.70 nmol/L、AEI为0.47；氯喹/赛庚啶的IC₅₀为81.53 nmol/L、AEI为0.37。5 nmol/L氯喹与625 nmol/L酮替芬（或赛庚啶）配伍，作用20 h，氯喹/酮替芬可使pfcrt基因表达水平升高91%，而氯喹/赛庚啶可使pfmdr1 基因表达水平下降14%。 结论 体外氯喹与适量的酮替芬（或赛庚啶）配伍，能增强恶性疟原虫氯喹抗性株对氯喹的反应性。氯喹对虫体作用 6～7 h 加入酮替芬（或赛庚啶）增效作用显著。该增效作用与pfcrt基因和pfmdr1 基因的表达水平有关。     

In vitro susceptibility of Plasmodium falciparum isolates from Myanmar to antimalarial drugs.

In vitro drug susceptibility profiles were assessed in 75 Plasmodium falciparum isolates from 4 sites in Myanmar. Except at Mawlamyine, the site closest to the Thai border, prevalence and degree of resistance to mefloquine were lower among the Myanmar isolates as compared with those from Thailand. Geometric mean concentration that inhibits 50% (IC50) and 90% (IC90) of Mawlamyine isolates were 51 nM (95% confidence interval [CI], 40-65) and 124 nM (95% CI, 104-149), respectively. At the nearest Thai site, Maesod, known for high-level multidrug resistance, the corresponding values for mefloquine IC50 and IC90 were 92 nM (95% CI, 71-121) and 172 nM (95% CI, 140-211). Mefloquine susceptibility of P. falciparum in Myanmar, except for Mawlamyine, was consistent with clinical-parasitological efficacy in semi-immune people. High sensitivity to artemisinin compounds was observed in this geographical region. The data suggest that highly mefloquine-resistant P. falciparum is concentrated in a part of the Thai-Myanmar border region.