青蒿琥酯联合克林霉素延缓恶性疟抗药性的实验研究

目的了解克林霉素与青蒿琥酯联用对抗青蒿琥酯恶性疟原虫是否有增效和延缓抗性作用。方法将恶性疟原虫用Trager法进行体外连续培养,待其正常生长后分组进行药敏试验。对照组单用青蒿琥酯处理,实验组用青蒿琥酯/克林霉素处理,然后在相同的培养时间内对两组虫株用相应的药物间断增加浓度进行刺激培育,在培育前及用药后不同时间用Rieckmann体外微量法测定其半数抑制量（ID50）。结果用药前青蒿琥酯和克林霉素对恶性疟原虫的ID50分别为14.4和14 610.4 nmol/L,两药联用时的ID50分别为青蒿琥酯6.4 nmol/L,克林霉素101.8 nmol/L。用药后90 d,对照组青蒿琥酯和克林霉素的ID50分别为273.8和24 815.2 nmol/L,实验组分别是49.6和23 859.4 nmol/L;停药后30 d对照组青蒿琥酯和克林霉素的ID50分别为155.8和21 543.2 nmol/L;实验组分别是32.0和15 487.4 nmol/L。结论青蒿琥酯和克林霉素联用有增效作用,能减缓恶性疟原虫对青蒿琥酯抗药性的产生速度。     

云南省恶性疟原虫抗青蒿琥酯株的体外培养育

为建立抗青蒿琥酯恶性疟原虫虫株,将采自恶性疟病人血,用Trager法进行体外连续培养,待其正常生长后,在培养基中加不同浓度的青蒿琥酯进行培育,并在培育前及用药后不同时间用Rieckmann体外微量法测定青蒿琥酯半数抑制量(ID50).用药前及用药后68 d、129 d及停药后67 d的ID50分别为9.6、30.6、85.1及52.9 nmol/L.结果表明可用人工方法建立高度抗青蒿琥酯恶性疟原虫虫株,停药后抗性程度有所下降.     

中缅边境西段缅甸恶性疟原虫对氯喹、青蒿琥酯及咯萘啶的敏感性体外测定

应用Rieckmann体外微量法测得中缅边境西段缅甸境内感染的恶性疟原虫对氯喹及我国抗疟新药青蒿琥酯及咯萘啶的抗性率分别为100％、14.3％及19.0％,半数抑制量(ID_(50)依次为249.5、4.2及12.1nmol/L。3例抗青蒿琥酯恶性疟原虫对氯喹和咯萘啶的ID_(50)分别为335.6nmol/L和43.1nmol/L;4例抗咯萘啶恶性疟原虫对氯喹和青蒿琥酯的ID_(50)分别为260.1nmol/L和5.0nmol/L。结果显示抗青蒿琥酯恶性疟原虫对咯萘啶及氯喹有明显的交叉抗性;抗咯萘啶恶性疟原虫对青蒿琥酯无交叉抗性。当地恶性疟原虫对氯喹的抗性程度明显高于云南南部恶性疟原虫,但对其它2种药物的敏感性则无明显差别,提示当地恶性疟原虫对青蒿琥酯及咯萘啶无交叉抗性。     

青蒿琥酯与萘酚喹联用延缓恶性疟原虫抗性实验研究

目的 探讨青蒿琥酯与萘酚喹联用是否能延缓恶性疟原虫抗性。方法 用青蒿琥酯与萘酚喹联用(A组)和单用青蒿琥酯(B组)间断刺激体外连续培养的恶性疟原虫，在接触药物前后不同时间用Reickmann体外微量测定恶性疟原虫的敏感性，同时观察每次接触药物后恶性疟原虫恢复正常生长时间。结果 A组用药前及用药后65d青蒿琥酯／萘酚喹的ID50分别为2．42／37．81、1．70／26．73nmol／L。B组用药前及用药后68、129d青蒿琥酯的ID50分别为9．60、30．61nmol／L和85．10nmol／L。A组接触药物后恶性疟原虫第1、2次恢复正常生长时间分别为24、37d，第3次接触药物后连续观察90d，疟原虫未能恢复正常生长。B组疟原虫平均恢复正常生长时间为16．7d。结论 可用体外间断药物刺激培育抗青蒿琥酯恶性疟原虫；青蒿琥酯与萘酚喹联用能有效延缓恶性疟原虫抗性。     

青蒿琥酯敏感与抗性株恶性疟原虫对本芴醇、蒿甲醚、双氢青蒿素的体外敏感性

为了解抗青蒿琥酯恶性疟原虫对本芴醇、蒿甲醚、双氢青蒿素是否存在交叉抗性 ,本芴醇与青蒿琥酯伍用是否有增效作用 ,运用 Rieckmann体外微量法测定。结果上述 3种药物对青蒿琥酯敏感株恶性疟原虫的 ID5 0 分别为 72 .3、14.6及 13.4nm ol/ L ;对抗性株的 ID5 0 依次为 6 6 .8、80 .5及 72 .6 nmol/ L。蒿甲醚、双氢青蒿琥酯对抗性株的 ID5 0 分别较敏感株高出 4.5及 4.4倍 ,本芴醇对敏感株的 ID5 0 与抗性株相似。在本芴醇与青蒿琥酯伍用中 ,两者对抗性株的 ID5 0 分别为 44 .8及 39.6 nmol/ L ,为单用组的 1/ 1.5 4(44 .7/ 6 6 .8)和 1/ 2 .2 (39.6 / 85 .1)。结果提示抗青蒿琥酯恶性疟原虫对本芴醇无交叉抗性 ,青蒿琥酯与本芴醇伍用在体外测定中具有一定增效作用 ;抗青蒿琥酯恶性疟原虫对蒿甲醚、双氢青蒿素有明显的交叉抗性     

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。     

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.     

云南省恶性疟原虫抗青蒿琥酯株的体外培养选育

为建立抗青蒿琥酯恶性疟原虫虫株 ,将采自恶性疟病人血 ,用 Trager法进行体外连续培养 ,待其正常生长后 ,在培养基中加不同浓度的青蒿琥酯进行培育 ,并在培育前及用药后不同时间用 Rieckmann体外微量法测定青蒿琥酯半数抑制量 (ID5 0 )。用药前及用药后 6 8d、12 9d及停药后 6 7d的 ID5 0 分别为 9.6、30 .6、85 .1及 5 2 .9nm ol/L。结果表明可用人工方法建立高度抗青蒿琥酯恶性疟原虫虫株 ,停药后抗性程度有所下降     

恶性疟原虫二氢乳清酸脱氢酶抑制剂体外诱导恶性疟原虫耐药的实验研究

目的分析恶性疟原虫二氢乳清酸脱氢酶(Plasmodium falciparum dihydroorotate dehydrogenase,PfDHODH)抑制剂(二氢噻吩酮类化合物,编号50,以下简称PfDHODH抑制剂50)对体外培养恶性疟原虫的作用特点及其诱导耐药的可能机制。方法恶性疟原虫氯喹敏感株(3D7株)和氯喹抗性株(Dd2株)同步化培养后分为不加药对照组、环状体期加药组和大滋养体期加药组,药物终浓度为80 nmol/L。分别在同步化后0 h(环状体期)、24 h(大滋养体期)、42 h涂薄血膜片镜检;通过逐步加大药物浓度的方法,体外诱导产生耐药虫株,3个月后经有限稀释培养,获得单克隆耐药虫株。采用SYBR GreenⅠ染料法检测各耐药虫株对PfDHODH抑制剂50、氯喹和青蒿素的半数抑制浓度(IC_(50));PCR扩增各耐药虫株Pfdhodh基因并测序,分析其突变情况。结果与不加药对照组相比,环状体期加药组恶性疟原虫从滋养体到裂殖体的发育受到明显抑制,大滋养体期加药组恶性疟原虫呈现明显的空泡化,核质密度大大降低。通过体外诱导并经有限稀释培养,获得44株PfDHODH抑制剂50的单克隆耐药虫株,其中,母本为Dd2、3D7的耐药虫株分别为24和20株,它们对PfDHODH抑制剂50的IC_(50)分别为(2.284±0.096)和(0.678±0.018)μmol/L,较母本虫株的(0.018±0.002)和(0.015±0.002)μmol/L分别提高了近130倍和50倍;对氯喹和青蒿素的IC_(50)分别为(0.011±0.002)、(0.014±0.004)和(0.013±0.003)、(0.012±0.001)μmol/L;与母本Dd2虫株相比,Dd2耐药虫株对氯喹的IC_(50)从(0.072±0.002)μmol/L下降为(0.011±0.002)μmol/L。测序分析结果显示,23株Dd2来源的耐药虫株PfDHODH蛋白氨基酸序列发生了G181D的点突变,另有1株除G181D的点突变外,还产生了K32N的点突变;3D7来源的耐药虫株未发现相应突变。结论体外诱导获得PfDHODH抑制剂50的单克隆耐药虫株,G181D的点突变可能是导致恶性疟原虫高水平耐受PfDHODH抑制剂50的重要分子机制。     

云南南部恶性疟原虫对氯喹、咯萘啶、青蒿琥酯、哌喹敏感性的体外测定

1988及1990年在滇南恶性疟流行区用体外微量法测得恶性疟原虫对氯喹、咯萘啶、青蒿琥酯及哌喹的抗性率分别为98.7％(75/76)、27.6％(16/58)、13.8％(9/65)及97.7％(43/44);半数抑制量(ID_(50))分别为125.0、19.0、4.7及243.3nmol/L,抗氯喹恶性疟原虫对哌喹有明显的交叉抗性;对咯萘啶及青蒿琥酯则未见明显的交叉抗性。抗青蒿琥酯恶性疟原虫对以上3种药均有一定程度的交叉抗性。抗咯萘啶恶性疟原虫对青蒿琥酯无交叉抗性,而对氯喹及哌喹有一定程度的交叉抗性。     

Drug-resistant malaria in Bangladesh: an in vitro assessment

Forty-four Plasmodium falciparum isolates from Bangladesh and 22 from western Thailand were successfully tested for their drug susceptibility. High degrees of resistance were observed against chloroquine with geometric mean IC50s of 114.25 and 120.5 nM, respectively, for Bangladesh and western Thailand. Most isolates from both sites were sensitive to quinine, and all were sensitive to artesunate. Many isolates were considered in vitro resistant to mefloquine, but the geometric mean IC50 for the Thai isolates (98.79 nM) was 1.6 times (P = 0.002) higher than that of isolates from Bangladesh (60.3 nM). The high prevalence of in vitro mefloquine resistance in Bangladesh suggests that close surveillance is necessary to delay widespread multidrug resistant problems in the area.     

Effects of hydroxypyridinone iron chelators in combination with antimalarial drugs on the in vitro growth of Plasmodium falciparum

Using standard in vitro drug susceptibility methods, we assessed the antimalarial activity of 3 orally administered iron chelators (hydroxypyridinones) alone and in combination with conventional antimalarials drugs (quinine, mefloquine, artesunate, tetracycline, atovaquone) against a chloroquine-resistant Plasmodium falciparum isolate. When tested alone, all iron chelators and antimalarial compounds inhibited the growth of the parasites. IC50 values for iron chelators were 60-70 microM, whereas the IC50 values for antimalarial drugs were in nM ranges, with artesunate being the most potent. The derived isobolograms for the interaction of hydroxypyridinones and antimalarial drugs showed addition or mild antagonism, similar to desferroxamine (Sum of Fractional Inhibitory Concentration, sigma FIC < 0.5 or > 4.0). Despite the absence of synergy with conventional drugs, intrinsic antimalarial activity of hydroxypyridinones supports the continued assessment of these iron chelators as treatment adjuncts.     

Drug-resistance of Trypanosoma b. rhodesiense isolates from Tanzania

OBJECTIVE: To determine the drug resistance of Trypanosoma brucei rhodesiense strains isolated from sleeping sickness patients in Tanzania. METHOD: We first screened 35 T. b. rhodesiense strains in the mouse model, for sensitivity to melarsoprol (1.8, 3.6 and 7.2 mg/kg), diminazene aceturate (3.5, 7 and 14 mg/kg), suramin (5, 10 and 20 mg/kg) and isometamidium (0.1, 1.0 and 2 mg/kg). A 13 isolates suspected to be resistant were selected for further testing in vitro and in vivo. From the in vitro testing, IC(50) values were determined by short-term viability assay, and MIC values were calculated by long-term viability assay. For in vivo testing, doses higher than those in the initial screening test were used. RESULTS: Two T. b rhodesiense stocks expressed resistance in vivo to melarsoprol at 5 mg/kg and at 10 mg/kg. These strains had high IC(50) and MIC values consistent with those of the melarsoprol-resistant reference strain. Another isolate relapsed after treatment with 5 mg/kg of melarsoprol although it did not appear resistant in vitro. One isolate was resistant to diminazene at 14 mg/kg and another was resistant at both 14 and 28 mg/kg of diminazene. These two isolates had high IC(50) values consistent with the diminazene-resistant reference strain. Two isolates relapsed at a dose of 5 mg/kg of suramin, although no isolate appeared resistant in the in vitro tests. Two isolates were resistant to isometamidium at 1.0 mg/kg and had higher IC(50) values. Two isolates were cross-resistant to melarsoprol and diminazene and one isolate was cross-resistant to suramin and isometamidium. CONCLUSION: The reduced susceptibility of T. b. rhodesiense isolates to these drugs strongly indicates that drug resistance may be emerging in north-western Tanzania.     

In vitro monitoring of Plasmodium falciparum susceptibility to artesunate, mefloquine, quinine and chloroquine in Cambodia: 2001-2002

We used a classical isotopic microtest to assess the in vitro sensitivity of 352 Plasmodium falciparum isolates collected in Cambodia in 2001 and 2002 to chloroquine, mefloquine, quinine and artesunate. Our results confirm conclusions drawn from earlier studies conducted by the Cambodian national malaria centre. Chloroquine-resistant phenotypes were highly prevalent in Cambodia. Similarly, a high proportion of isolates displayed elevated IC50 to mefloquine. In contrast, only 0.67 and 1.7% of isolates presented decreased susceptibility to quinine and artesunate, respectively. Distributions of mean IC50 according to drug and geographic origin indicated that the parasites circulating to the west of Cambodia largely account for the global situation of drug resistances in Cambodia. Isolates with decreased susceptibility to chloroquine and mefloquine were common along the border with Thailand. In contrast, most of the isolates from eastern Cambodia were susceptible to these compounds. Isolates collected at the western and eastern borders did not respond differently to artesunate. No major differences in responses to antimalarial drugs were observed between 2001 and 2002, suggesting that the situation of drug resistance is now stabilized and under control in Cambodia. However, the decreased susceptibility of isolates collected in the western provinces of Cambodia to mefloquine and the correlation between susceptibility to artesunate and susceptibility to mefloquine and quinine justify the need for an improved international surveillance program for malaria drug resistance in the Mekong sub region.     

咯萘啶、甲氟喹和奎宁对青蒿琥酯敏感株与抗性株恶性疟原虫的体外作用

[目的 ]比较青蒿琥酯敏感株与抗性株恶性疟原虫对咯萘啶、甲氟喹和奎宁的敏感性 ;了解这 3种药物与青蒿琥酯伍用对抗青蒿琥酯恶性疟原虫的体外抗疟作用。 [方法 ]应用Rieckmann体外微量法测定敏感株与抗性株原虫对上述 4种药物的敏感性 ,同时测定青蒿琥酯分别与咯萘啶、甲氟喹、奎宁伍用对抗性株原虫的敏感性。 [结果 ]咯萘啶、甲氟喹、奎宁和青蒿琥酯对敏感株的ID5 0 分别为 5 9 0、 6 9 7、 2 83 8和 9 6nmo1/L ;对抗性株的ID5 0 依次为 170 6、 6 3 2、 2 72 4及 85 1nmo1/L。咯萘啶与青蒿琥酯伍用 ,其ID5 0 分别为两药单用组的 1/4 7(36 6 / 170 6 )和 1/ 3 7(2 2 8/ 85 1)。甲氟喹与青蒿琥酯伍用中 ,其ID95 分别为两药单用组的 1/ 4(40 / 16 0 )和 1/ 12 5 (3 2 / 40 0 )。奎宁与青蒿琥酯伍用 ,其ID95 分别为单用组的 1/ 16 (80 / 12 80 )和 1/ 12 5 (3 2 / 40 0 )。 [结论 ]抗青蒿琥酯恶性疟原虫对甲氟喹和奎宁无交叉抗性 ,这两种药物分别与青蒿琥酯伍用 ,对抗青蒿琥酯恶性疟原虫有较明显增效作用。     

中缅边境地区恶性疟原虫对7种抗疟药敏感性的体外测定

应用体外微量法检测恶性疟原虫对氯喹、氨酚喹、青蒿琥酯、还原青蒿素、蒿乙醚及咯萘啶的抗性率，我国境内的分别是９５．７％（２２／２３）、８８．９％（８／９）、１６．５％（４／２４）、２２．２％（２／９）、１１．１％（１／９）、２０．８％（５／２４）；半数抑制量（ＩＤ５０）分别为１７６、７２、６、１３、１１８、１６ｎｍｏｌ／Ｌ；ＩＤ９５依次为８３０、５１２、３４、８０、６５０、８８ｕｍｏｌ／Ｌ。缅甸境内的分别为１００％（２９／２９）、８３．３％（５／６）、１３．８％（４／２９）、１６．７％（１／６）、１６．７％（１／６）、２１．４％（６／２８）；ＩＤ５０分别为２４０、５２、６、１４、１２０、１８ｎｍｏｌ／Ｌ；ＩＤ９５为１４０８、３８４、３４、８０、６５０、９６ｎｍｏｌ／Ｌ。境内、外恶性疟原虫对奎宁均敏感，ＩＤ５０分别为６０８、４７０ｎｍｏｌ／Ｌ，ＩＤ９５为２５６０，１６９０ｎｍｏｌ／Ｌ。证实该地区恶性疟原虫对氯喹普遍存在高度抗性．且缅甸高于我国；对氨酚喹亦普遍产生抗性；对青蒿琥酯，还原青蒿素、蒿乙醚、咯萘啶有少数疟原虫产生低度抗性。抗氯喹恶性疟原虫对青蒿琥酯，还原青蒿素、蒿乙醇和咯萘啶无交叉抗性。抗青蒿琥酯恶性疟     

Anti-malarial efficacy of pyronaridine and artesunate in combination in vitro and in vivo

Pyronaridine is a Mannich base anti-malarial with demonstrated efficacy against drug resistant Plasmodium falciparum, P. vivax, P. ovale and P. malariae. However, resistance to pyronaridine can develop quickly when it is used alone but can be considerably delayed when it is administered with artesunate in rodent malaria models. The aim of this study was to evaluate the efficacy of pyronaridine in combination with artesunate against P. falciparum in vitro and in rodent malaria models in vivo to support its clinical application. Pyronaridine showed consistently high levels of in vitro activity against a panel of six P. falciparum drug-sensitive and resistant strains (Geometric Mean IC50=2.24 nM, 95% CI=1.20-3.27). In vitro interactions between pyronaridine and artesunate showed a slight antagonistic trend, but in vivo compared to pyronaridine and artesunate administered alone, the 3:1 ratio of the combination, reduced the ED90 of artesunate by approximately 15.6-fold in a pyronaridine-resistant P. berghei line and by approximately 200-fold in an artesunate-resistant line of P. berghei. Complete cure rates were achieved with doses of the combination above or equal to 8 mg/kg per day against P. chabaudi AS. These results indicate that the combination had an enhanced effect over monotherapy and lower daily doses of artesunate could be used to obtain a curative effect. The data suggest that the combination of pyronaridine and artesunate should have potential in areas of multi-drug resistant malaria.