Infection of Anopheles darlingi fed on patients with Plasmodium falciparum before and after treatment with quinine or quinine plus tetracycline

Anopheles darlingi fed on eight falciparum malaria patients with gametocytes before and after treatment with quinine sulfate or quinine sulfate plus tetracycline became infected. Quinine and quinine plus tetracycline had no apparent sporontocidal or gametocytocidal effect on late stage immature and mature gametocytes. Plasmodium falciparum gametocytes are persistent and infected mosquitoes for up to 21 days after patients were treated with quinine plus tetracycline. Sporogonic development was similar for groups of mosquitoes fed before and after patients were treated with these schizontocides. The percentages of infected mosquitoes that developed salivary gland infections were also similar for groups of mosquitoes fed before and after treatment. Twenty-four hours after treatment with 45 mg of primaquine phosphate, falciparum malaria patients were not infective to An. darlingi.     

Combination of the antibiotics erythromycin and tetracycline with three standard antimalarials against Plasmodium falciparum in vitro

Combinations of antibiotics and standard antimalarials have been assayed against P. falciparum in vitro, using incorporation of 14C isoleucine as an indicator of drug action. Chloroquine and erythromycin have been shown to act synergistically against a chloroquine-resistant strain and additively towards a chloroquine-sensitive strain, confirming their action against sensitive and resistant P. berghei in vivo, described elsewhere. Combinations of erythromycin with mefloquine or quinine acted anergically in 24 hour assays in which unphysiologically high concentrations of quinolinemethanol were necessary for demonstrable drug effect. In 48 hour assays, an additive effect was obtained with these combinations. Tetracycline is additive in combination with each of the standard antimalarials used in this study. The relevance of results obtained in vitro to parasite drug sensitivities in vivo is discussed.     

In vitro susceptibility of Plasmodium falciparum to quinine: relation to parasite density and drug distribution in culture fractions

We have studied the importance of parasite density (2, 0·2, 0·02 and 0·002%) for the in vitro susceptibility of Plasmodium falciparum (F32 strain) to quinine. Shorter exposures (48 hours) only briefly inhibited parasites in wells with the highest initial density. Parasites reappeared after 3–5.5 days in wells with intermediate (0·2 and 0·02%) and lowest density (0·002%). Longer exposures (72 hours), however, inhibited them for much longer periods and parasites did not reappear in most of the wells with the lowest density during the 28 days of follow-up. The mean multiplication rate following reappearance was tenfold per parasite schizogony cycle. The mean elimination rate per schizogony cycle was calculated to be 99·91%. The elimination and multiplication rates were not correlated to initial parasite density. The mean ratio between quinine concentrations in erythrocytes and medium was 3·6 regardless of quinine concentrations and presence of parasites. Mean quinine-free fractions of 36 and 67% were found from total concentrations of 0·33 and 10·4 µmol/1. We conclude that initial parasite density determines the time to reappearance of parasites following quinine exposure while the elimination and multiplication rates are independent of the initial parasite density, and that quinine protein binding is concentration-dependent in vitro and lower than during treatment.     

The in vivo and in vitro sensitivity of Plasmodium falciparum to quinine

The in vivo and in vitro sensitivity of P. falciparum to quinine were studied simultaneously on 20 isolates of P. falciparum from infected patients in Rangoon and in Tharrawaddy Township. The in vivo study showed 85% sensitive and 5% resistance at RI level. The peak plasma quinine level in all the cases were above mean MIC on days 1, 3, 5 and 7. Schizont maturation was inhibited at 128 p.mol/well in 15% of the cases but the rest were at or below 64 p.mol/well in vitro test. However, no relationship was detected between the in vivo and in vitro sensitivity of quinine.     

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.     

In vitro drug response of Plasmodium falciparum in the Philippines: increased resistance to amodiaquine

A long term study was carried out at San Lazaro Hospital, Manila, Philippines, monitoring the in vitro response of Plasmodium falciparum to chloroquine, amodiaquine, mefloquine, and quinine. The in vitro effective dose giving 50% inhibition of schizogony was: 0.68 X 10(-6) M/liter blood for chloroquine; 0.18 X 10(-6) for amodiaquine; 0.2 X 10(-6) for mefloquine; and 1.12 X 10(-6) for quinine. The percent of isolates determined to be resistant in vitro was 85.2% for chloroquine, and 1.2% for both mefloquine and quinine. These figures were relatively unchanged over the course of 3 years studied. The in vitro resistance rate to amodiaquine increased from 5.1% in 1982 to 22.2% in 1984.     

The effect of artemisinin combined with standard antimalarials against chloroquine-sensitive and chloroquine-resistant strains of Plasmodium falciparum in vitro

The activity of artemisinin (qinghaosu) in combination with some commonly-used antimalarial drugs was tested in vitro against a chloroquine-sensitive (NF54) and a chloroquine-resistant (K1) strain of Plasmodium falciparum. Both mefloquine and tetracycline showed marked potentiative synergism with artemisinin against both strains, whilst primaquine showed potentiation against K1. Combinations of artemisinin with pyrimethamine and with chloroquine were antagonistic. The results confirm observations on rodent malaria in vivo and indicate that the drug interactions seen were direct, through actions on the parasite, and not merely effects on drug distribution and metabolism in the mouse host.     

Plasmodium falciparum in vivo resistance to quinine: description of two RIII responses in French Guiana

The resistance of Plasmodium falciparum to antimalarial drugs is one of the most worrisome problems in tropical medicine, but few clinical studies or observations have described confirmed cases of therapeutic failure. We report two cases of in vivo P. falciparum resistance (RIII response) to quinine in French Guiana, an Amazonian focal zone in which multi-resistant malaria is endemic. Both patients presented with uncomplicated malaria and were initially treated with intravenous quinine. Although absorption was normal, the treatment was not effective and the patients still had fever and significant parasitemia three days after the onset of treatment (day 3). The addition of intravenous tetracycline completely resolved the parasitemia within approximately 96 hours. These clinical reports confirm the necessity to combine quinine with tetracycline in this area, as recommended by the recent French regional antimalarial policy.     

Decreased sensitivity to chloroquine and quinine of some Plasmodium falciparum strains from Senegal in September 1984

The in vitro sensitivity of 135 Plasmodium falciparum isolates collected in the areas of Thies and Kaolack, Senegal, were studied in September 1984, by means of the 48 hr microtest with 3H-hypoxanthine incorporation. Results are available in 110 of 135 cases studied (81%). The isolates collected around Kaolack were found on average less sensitive to chloroquine than those from Thies (mean EC50 28 and 17 nmol/l of medium, respectively, P less than 0.05) and in 1 isolate a high degree of resistance was found (EC50 178 nmol/l). Some of those isolates also were studied using the WHO standard microtest and similar results were recorded. With both assays probit regression lines show EC99 in Kaolack greater than 114 nmol/l. These data suggest that in 1984 chloroquine resistance was possibly emerging in the extreme west of Africa. Sensitivity to quinine could be evaluated in 15 of 24 isolates tested. One of them, originating from Thies, was highly resistant to this drug (EC50 760 nmol/l) and 2 additional isolates with EC50s of 370 and 274 nmol have decreased sensitivity. These results suggest that quinine, as well as chloroquine, sensitivity should be monitored in the African region.     

In vitro sensitivity of Plasmodium falciparum isolates from Burma to chloroquine quinine and mefloquine

The in vitro sensitivity of 26 isolates of Plasmodium falciparum from Rangoon and Tharrawaddy areas in Burma were studied on chloroquine, mefloquine and quinine. The results indicated that the parasites were highly resistant to chloroquine but sensitive to mefloquine and quinine. The existence of correlation of sensitivity to mefloquine and quinine was detected and discussed. No correlation between the parasite sensitivity to chloroquine and mefloquine and or chloroquine and quinine was detected.     

Patterns of in vitro resistance to chloroquine, quinine, and mefloquine of Plasmodium falciparum in Cameroon, 1985-1986

The drug sensitivity of 246 Plasmodium falciparum isolates was studied in vitro in five areas of Cameroon at the end of 1985. Results demonstrate that parasites resistant either to chloroquine, quinine, or mefloquine, or to two of these drugs, were prevalent in four of the areas investigated, but the drug response pattern varies widely from one area to another. The recent explosive emergence of chloroquine resistance in the south of the country, where both prevalences and levels are very high (up to 86%), contrasts with only moderate levels of resistance in the north. This may be related to differences in transmission by mosquitoes between Sahel and forest areas. Quinine resistance was observed in 24% of the isolates studied in vitro and was frequently associated with chloroquine resistance. The presence of isolates responding poorly to mefloquine, observed mainly in northern Cameroon, suggests that resistance may occur in areas where the drug has never been used.     

缅甸拉咱市恶性疟原虫对6种抗疟药的敏感性体外测定

目的了解中缅边境缅甸拉咱市恶性疟药物抗性现状,为中国第六轮全球基金疟疾项目制定防治策略提供依据。方法在缅甸拉咱市城区建立研究站,镜检发热病人和/或疑似疟疾病例,筛查疟疾患者。选择城区及郊区4村的单纯恶性疟现症患者,用WHO推荐的体外微量试验测定恶性疟原虫对6种抗疟药的敏感性。结果体外微量测定43例,成功27例,测得恶性疟原虫对氯喹、哌喹、咯萘啶、青蒿琥酯、双氢青蒿素和双氢青蒿素/哌喹的抗性率分别为100%、0、25.9%、22.2%、7.4%和0,半数抑制浓度(IC50)分别为298.0、49.4、22.3、24.3、11.8和5.6/44.8nmol/L。结论缅甸拉咱市流行株恶性疟原虫对氯喹100%抗性,对咯萘啶、青蒿琥酯和双氢青蒿素具有一定程度抗性,对氯喹、咯萘啶和青蒿琥酯抗性呈逐渐增强趋势,对哌喹和双氢青蒿素/哌喹敏感。     

In vitro evaluation of quinidine sensitivity in Brazilian Plasmodium falciparum isolates: comparative analysis to quinine and chloroquine

Falciparum malaria represents a serious and an increasing world public health problem due to the acquired parasite's resistance to the most available drugs. In some endemic areas, quinidine, a diastereoisomer of the antimalarial quinine, has been employed for replacing the latter. In order to evaluate the use of quinidine as an alternative to the increasing loss of quinine effectiveness in Brazilian P. falciparum strains, as has been observed in the Amazon area, we have assayed quinidine, quinine and chloroquine. The in vitro microtechnique was employed. All isolates showed to be highly resistant to chloroquine. Resistance to quinine was not noted although high MIC (minimal inhibitory concentration) values have been observed. These data corroborate the decreasing sensitivity to quinine in strains from Brazil. Quinidine showed IC50 from 0.053 to 4.577 micromol/L of blood while IC50 from 0.053 to 8.132 micromol/L of blood was estimated for quinine. Moreover, clearance of the parasitemia was observed in concentrations lower than that used for quinidine in antiarrhythmic therapy, confirming our previous data. The results were similar to African isolate.     

In vitro blood schizonticidal activity of sera containing mefloquine‐quinine against Plasmodium falciparum

Serum samples collected at intervals from healthy volunteers, after the administration of 3 drug regimens (quinine (QN) 600 mg, mefloquine (MQ) 750 mg, and MQ 750 mg plus QN 600 mg) were investigated for their blood schizonticidal activities against K₁ strain Plasmodium falciparum in vitro . Superiority of activity was shown in the sera collected after the combination regimen. In the diluted sera of the QN regimen, a complete inhibitory effect was observed for only 24 hours, whereas the effect was sustained for 72 hours in the sera collected after MQ in either regimen (MQ alone or MQ/QN). The pattern of minimum inhibitory concentrations (MICs) of QNEq of the sera from QN alone was constant throughout a 24‐hour period, with significantly higher concentrations than that from the combination regimen (118–150 vs 21.25–73.5 μg/l). In sera collected after the combination regimen, however, the MIC gradually decreased from 0.5 until 2.5 and 4 h, and thereafter gradually returned to the same levels again during a period of 6–24 hours. The MICs of MQEq when given as MQ alone or in combination appeared constant, with a significantly higher value in the former regimen (24.4–26.8 vs 17–19.2 μg/l).     

Molecular assessment of Plasmodium falciparum resistance to antimalarial drugs in China

Objective  In China, Chloroquine (CQ) and sulfadoxine–pyrimethamine (SP) were abandoned for the treatment of falciparum malaria 20 years ago due to resistance. Subsequent field studies showed a trend of declining CQ and SP resistance in the country. The main purpose of this study was to analyse the molecular markers of antimalarial resistance and thereby to assess the possibility of reintroduction of CQ or SP for falciparum malaria treatment.
Methods  Plasmodium falciparum field isolates were collected in 2006–2007 from Hainan and Yunnan provinces, China. Nested PCR-sequencing assays were applied to analyse the SNPs in four genes: P. falciparum chloroquine resistance transporter ( pfcrt ) gene, multi-drug resistance 1 ( pfmdr1 ) gene, dihydrofolate reductase ( dhfr ) gene and dihydropteroate synthetase ( dhps ) gene.
Results  We found the widespread presence of point mutations in the dhfr and dhps genes which are associated with SP treatment failure. The molecular analyses also showed the fairly high prevalence of point mutation in the pfcrt gene which is linked to CQ resistance.
Conclusion  The results of the present study indicate that CQ and SP should not be reintroduced for falciparum malaria treatment in the near future in China.     

In vitro activity of chloroquine and quinine in combination with desferrioxamine against Plasmodium falciparum

The activity of chloroquine and quinine, alone and in combination with desferrioxamine (7 μmol/liter), was evaluated in vitro against susceptible and resistant clones of Plasmodium falciparum by a semimicroassay system. The addition of desferrioxamine had no effect on the activity of chloroquine against both clones. Desferrioxamine had no effect on the activity of quinine against the susceptible clone but had slightly enhanced quinine action against the resistant clone. Further development of desferrioxamine as an antimalarial drug may be of limited interest. © 1993 Wiley-Liss, Inc.     

Mefloquine sulfadoxine pyrimethamine (MSP) combination delays in vitro emergence of mefloquine resistance in multiple drug resistant Plasmodium falciparum

Sulfadoxine-pyrimethamine-resistant and chloroquine resistant, but mefloquine sensitive (MIC 5 x 10(-9] isolates of Plasmodium falciparum were used to study the emergence of mefloquine resistance. The continuously cultured isolates which were exposed intermittantly to varying concentrations of mefloquine alone became insensitive to the drug at 1 x 10(-7) M after 12 months, whereas the culture line exposed to the combination of mefloquine, sulfadoxine and pyrimethamine became only slightly insensitive to mefloquine (MIC 2 x 10(-8]. Thus, the efficacy of mefloquine may be prolonged through use in combination with sulfadoxine-pyrimethamine.     

In vivo and in vitro studies of quinine sensitivity of Plasmodium falciparum in Thailand.

Forty-four patients with falciparum malaria were studied. Nine patients were given quinine orally at a daily dose of 1.5 gm base for a period of 14 days. The mean parasite clearance in all 9 patients was 3.3 days, and none had recrudescence in follow-up examinations for 31 days. The in vivo study of these 9 patients showed sensitivity to quinine which correlated with the in vitro test, with concentration of quinine base 2.5-5.8 microgram/ml of blood that inhibited the maturation of Plasmodium falciparum parasites. The results of the in vitro test of 35 patients showed concentrations of quinine base 2.1-5.4 microgram/ml of blood were able to inhibit the maturation of P. falciparum parasites. Therefore, these studies indicate that Plasmodium falciparum are still sensitive to quinine and quinine remains to be the drug of choice for the treatment of falciparum malaria in Thailand.