Chloroquine-resistant Plasmodium falciparum with reduced sensitivity in vitro to mefloquine and quinine in Zaria, northern Nigeria

Thirty-six P. falciparum isolates collected from children with malaria were tested for their susceptibility to chloroquine, mefloquine and quinine in vitro using the WHO microtest system. 37% of the isolates grew in the presence of 1.6 mumol chloroquine 1(-1) blood, indicating resistance. The sensitivity to both mefloquine and quinine was markedly reduced. The inhibitory endpoints for quinine correlated with those for chloroquine and mefloquine, but no such correlation was found between chloroquine and mefloquine.     

Micro in vitro assessment of Plasmodium falciparum sensitivity to chloroquine and mefloquine in Gujarat

Micro in vitro tests conducted in 1987 in Surat district of Gujarat on sensitivity status of P. falciparum to chloroquine and mefloquine revealed that the parasite has developed resistance to chloroquine upto 32 pmol. The ED 99 in Hazira, Gothan and Umra areas of the district was found to be 17.3, 18.5 and 8.7 pmol/well for chloroquine and for mefloquine it was 14.5, 4.8 and 6.8 pmol/well respectively. Monitoring of P. falciparum resistance is indicated under National Malaria Eradication Programme.     

Efficacy of artemisinin and mefloquine combinations against Plasmodium falciparum. In vitro simulation of in vivo pharmacokinetics

The activity of artemisinin in combination with mefloquine was tested in vitro against a chloroquine‐sensitive (F32) strain of Plasmodium falciparum . A method of repetitive dosing and extending the culture observation period to 28–30 days was used to mimic the in vivo pharmacokinetic situation. Plasmodium falciparum was exposed to artemisinin from 10⁻⁸ to 10⁻⁵  m , mefloquine from 3×10⁻⁹ to 10⁻⁵  m and their combinations. The exposure time for artemisinin was 3 hours twice daily and for mefloquine 24 hours. The drug‐dosing duration was 3 days.
Neither artemisinin nor mefloquine alone provided radical clearance of P. falciparum , even when maximum concentrations (10⁻⁵  m ) were applied. The antiparasitic activity of artemisinin and mefloquine were significantly higher when dosed alone. Effective concentrations for different degrees of inhibition (EC 50, 90 and 99) of both artemisinin and mefloquine respectively were significantly lower when used in combination. At concentrations normally reached in vivo , this effect was clearly synergistic ( P =0.016)
Our in vitro model of intermittent dosing of artemisinin and mefloquine combinations for 3 days provides significant evidence of positive interaction between the two compounds. Lower combination concentrations around the MIC‐values for the individual compounds showed synergistic effect, and high concentrations showed additive effect. This indicates that such drug combinations may provide radical clearance at concentrations lower than those required for single‐drug treatment.     

Fluoxetine potentiates chloroquine and mefloquine effect on multidrug-resistant Plasmodium falciparum in vitro

Fluoxetine (FLX), a P-glycoprotein inhibitor with antimalarial activity, is promising candidate for reversing chloroquine/mefloquine (CQ/MQ) resistance. The Dd2 strain of CQ- and MQ-resistant Plasmodium falciparum was synchronized and assayed with various concentrations of CQ/MQ individually and in combination with FLX or verapamil (VPL). Our results indicated the 50% inhibitory concentration values of CQ and MQ were greatly lowered when FLX was used simultaneously. Isobolograms indicated that CQ-FLX combinations are more synergistic (mean fractional inhibitory concentration [FIC] index 0.55) than MQ-FLX (mean FIC index 0.64), and their synergistic effect was better than or at par with CQ-VPL (mean FIC index 0.88) and MQ-VPL (mean FIC index 0.60) combinations. We conclude that the FLX potentiates the CQ and MQ response on multidrug-resistant P. falciparum at clinically achievable concentrations.     

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.     

In vitro and clinical correlates of mefloquine resistance of Plasmodium falciparum in eastern Thailand

A series of isolates of Plasmodium falciparum from eastern Thailand was collected prior to and after treatment failure with mefloquine. Patterns of drug sensitivity to standard and new antimalarials were characterized by using an in vitro assay based on the inhibition of schizont maturation. In vitro levels of mefloquine sensitivity of isolates were correlated with clinical treatment failures. In vitro parasite resistance to mefloquine is defined as an inhibitory dose-50 value greater than 20 nM. For isolates collected prior to treatment, there was no significant difference in mefloquine sensitivity patterns between subsequent successes and failures, suggesting that mefloquine treatment failures could not be predicted based on in vitro sensitivity of pretreatment isolates. A series of paired isolates were collected both prior to treatment with mefloquine and after recrudescence. Recrudescent isolates showed significant decreases in sensitivity to mefloquine, WR 194965, enpiroline, and halofantrine; no significant changes in sensitivity to amodiaquine, qinghaosu, and pyrimethamine; and an increase in sensitivity to chloroquine.     

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.     

微量荧光法体外测定恶性疟原虫对4种常用抗疟药的敏感性

目的 验证微量荧光法(MFA)用于恶性疟原虫体外药物敏感性和抗疟药物筛选的适用性。方法 运用MFA测定氯喹、青蒿素、蒿甲醚和咯萘啶等4种常用抗疟药物对体外培养FCC1/HN株恶性疟原虫的敏感性,并对该方法所测得的量效曲线(dose-response curves)及50%有效抑制浓度(IC₅₀)与光学显微镜镜检法所得的结果进行比较。结果 MFA所测定的氯喹、青蒿素、蒿甲醚和咯萘啶的IC50分别为18.79、6.32、3.67和2.00 nmol/L,光学显微镜镜检法的结果分别为19.65、5.82、4.38和2.83nmol/L,两者差异均无统计学意义(P>0.05)。结论 用MFA体外测定恶性疟原虫对抗疟药的敏感性敏感、快速,可用于体外抗疟药物的敏感性测定及抗疟药物筛选。     

Responses of multidrug-resistant Plasmodium falciparum parasites to mefloquine in Nigerian children

Summary Thirty-three children aged 6 months to 7 years from an area with multidrug-resistant Plasmodium falciparum strains were treated with 25 mg/kg body weight of mefloquine base as a single oral dose. They were followed-up using the modified 28-day WHO extended field test. The parasite isolates from these patients were cultured in vitro with different concentrations of mefloquine. All children were parasite-negative by day 4, and 31 remained so throughout the period of observation. Two patients who were parasitaemic on days 16 and 28 were successfully treated with a sulphadoxine/pyrimethamine combination. Parasitological and clinical responses were well correlated. The mean parasite clearance time was 65 ± 10.2 hours. A mefloquine concentration of 64 pmol/well inhibited schizont growth and the EC₅₀ and EC₉₉ were 5.5 and 54 pmol/well (1.1 and 10.8 μmol/l blood) respectively. This indicates reduced parasite susceptibility to the drug in vitro .     

Correlation between in vivo and in vitro response of chloroquine-resistant Plasmodium falciparum in Calabar, south-eastern Nigeria.

Since chloroquine-resistant Plasmodium falciparum (CRPF) has emerged in Nigeria, we monitored the susceptibility of the parasite strain to a standard chloroquine (C25) dose in our Children's Emergency Unit. Chloroquine (CQ) is the drug of choice for malaria chemotherapy in Nigeria. The WHO 7-day in vivo evaluation and Rieckmann's microtitre technique (in vitro test) were used. 33 children of mean age 4.9 years were enrolled in the study. 27 (81.8%) of the in vitro cultures were successful. 16 (59.3%) of the successful isolates still showed schizogony at CQ concentration of 5.7 pmol/well and above. 28 (84.8%) of the children completed the in vivo study. 15 (53.6%) were parasitaemic on day 7 and/or day 14 and were regarded as parasitologic failures. The isolates from 14 of these children showed corresponding in vitro resistance of CQ concentrations equal to or above 5.7 pmol/well. The proportion of RIII (= 13.3%) appears to have increased as compared to 5.9% recorded in 1987. We conclude that there appears to be a good correlation between in vivo evaluation of parasitologic failures (53.6%) and in vitro resistance (59.3%). It thus appears that CRPF is definitely increasing in South-Eastern Nigeria. This can be expected not only to complicate malaria chemotherapy in the Children's Emergency Unit of the University of Calabar Teaching Hospital, but will contribute immensely to the deterioration of malaria therapy and control in Nigeria.     

Efficacies of mefloquine alone and of artesunate followed by mefloquine, for the treatment of uncomplicated, Plasmodium falciparum malaria in eastern Sudan

In late 2003, the efficacies of mefloquine monotherapy and of an artesunate-mefloquine combination, for the oral treatment of uncomplicated, Plasmodium falciparum malaria, were investigated and compared in New Halfa, in eastern Sudan. Of the patients who completed the 28 days of follow-up, 40 were treated only with single-dose mefloquine (at a dose of 25 mg/kg), and 38 with artesunate (at 4 mg/kg. day) for 3 days followed by single-dose mefloquine (at 15 mg/kg), given on the third day.Compared with those given the combination, the patients given mefloquine alone were more likely to suffer nausea, vomiting and dizziness (25.0% v. 2.6%; P=0.005) and to be found gametocytaemic (12.5% v. 0%; P=0.02) after treatment, and more likely to be found febrile (i.e. with a temperature >37.5 degrees C) on day 2 (25.0% v. 2.6%; P=0.005), although no patients were found febrile on day 3. Six of the patients--three (7.5%) of those given mefloquine only and three (7.9%) of those given the combination (P>0.05)--appeared to be treatment failures. Parasite genotyping indicated, however, that, although five of these six patients had true recrudescences, one (who had been treated with the combination) had been re-infected during the follow-up. The true frequencies of cure were therefore 92.5% after mefloquine alone and 94.7% after the combination (P>0.05). Thus, although the treatments appeared equally effective in clearing parasitaemias, the combination was better at clearing gametocytaemias and was less likely to cause adverse side-effects. It remains unclear why mefloquine given alone was almost 10-fold more likely to trigger adverse effects than treatment with a combination that contained the same drug. This may be a reflection of the different mefloquine doses and, for the patients given the combination, of the use of artesunate before the mefloquine treatment.     

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 vitro susceptibility of Plasmodium falciparum isolates to chloroquine and mefloquine in southeastern Mindanao Island, the Philippines

Although the presence of multi-drug-resistant falciparum malaria has been reported in the Philippines, the distribution of drug-resistant malaria parasites has not yet been determined in Mindanao Island. In vitro susceptibility of P. falciparum to both chloroquine and mefloquine was assessed to forecast the spread of drug-resistant parasites in various foci in southeastern Mindanao Island. Of the 33 isolates of P. falciparum successfully tested, 10 (30%) were susceptible, 12 (36%) showed decreased susceptibility (80 nM < or = IC50 < 114 nM), and 11 (33%) were resistant (IC50 > or = 114 nM) to chloroquine. Ten (91%) of the resistant isolates and 9 (75%) of those with decreased susceptibility were from northern and northwestern Davao del Norte Province. Chloroquine-susceptible isolates were found among patients in the eastern parts of Davao del Norte and Davao Oriental provinces. Seven isolates from several foci in the study area were all mefloquine- susceptible (IC50 < 10 nM). This is the first report indicating the potential emergence of chloroquine-resistant P. falciparum on Mindanao Island, which is presently regarded as a drug-susceptible area.     

一种便于现场测定恶性疟原虫抗药性的培养基

为了各地开展体外微量测定法调查恶性疟原虫的抗药性,研制了便于现场调查使用的培养基。将连续培养恶性疟原虫用的RPMI1640完全液体培养基用安瓿封装,冰瓶贮存.可供现场50d内使用。该培养基的效果与冰冻干燥培养基相似,明显优于WHO培养基。     

Formation of Plasmodium falciparum gametocytes in vivo and in vitro relates to transmission intensity

Plasmodium falciparum isolates were collected during a cross-sectional survey of fever cases in an endemic area of northern India, over a period of 6 months (July-December 1996). Smears of peripheral blood from 118 cases were found to contain Plasmodium falciparum. Isolates of the parasites were successfully cultured in vitro, under identical conditions, from blood samples from 58 of the smear-positives who had asexual parasitaemias of at least 0.05%. Gametocytes developed from 50 of the 58 isolates, with a variable range of gametocytaemia. An isolate was most likely to produce gametocytes and to produce high gametocytaemias (i.e. > or = 1.0%) in vitro if it had been collected when the intensity of transmission (as reflected in bloodsmear positivity) was relatively high. A young child (< 6 years) with fever was more likely to be smear-positive for the asexual and sexual stages of P. falciparum, but less likely to be seropositive for antibodies to these stages, than the older cases of fever.     

Plasmodium falciparum in Madagascar: in vivo and in vitro sensitivity to seven drugs

The sensitivity level of Plasmodium falciparum isolates to chloroquine and the activity of six other antimalarials were studied in the different climatic zones of Madagascar in 1983. In vivo tests were done with 10 and 25 mg kg-1 of chloroquine and amodiaquine. Early recrudescence or RII resistance was observed after treatment with 10 mg kg-1 of these drugs in 34% of the cases for chloroquine and 6.5% for amodiaquine, and after the 25 mg kg-1 dose in 7% and 0% of the cases respectively. In vitro sensitivity of 84 P. falciparum isolates to seven drugs were studied with a semi-microtest. For chloroquine, 9% of the isolates had an IC50 above 250 nM, indicating resistance. In vitro activity of piperaquine was high for all isolates except two. In vitro activity of amodiaquine, dichlorquinazine, quinine, mefloquine and halofantrine was good against all isolates (maximum IC50 was 76, 92, 560, less than or equal to 20 and less than or equal to 12 nM, respectively). Correlation between the WHO standard field test and the in vitro semi-microtest was good. Resistance of P. falciparum to chloroquine was observed in the six survey areas, but the other tested drugs showed good activity. Since no cross-resistance to 4-aminoquinolines seems to exist in Madagascar, amodiaquine (the only one available at present) should be studied as an alternative to chloroquine in the prevention and treatment of falciparum malaria in this area.     

In vitro mefloquine resistance of Plasmodium falciparum isolated from the Burmese border region of Thailand

The in vitro susceptibility of twenty isolates of Plasmodium falciparum from Tha Song Yang, Tak province, Thailand were determined. The isolates were resistant to chloroquine (IC50 = 220 nM; MIC = 762 nM), quinine (IC50 = 252 nM; MIC = 1010 nM), and pyrimethamine (IC50 = 16400 nM; MIC = 43100 nM) but generally sensitive to mefloquine (IC50 = 6.90 nM; MIC = 20.9 nM) and halofantrine (IC50 = 8.73 nM; MIC = 2.71 nM). Two isolates were identified which appeared resistant to mefloquine (IC50 = 23.1 nM; MIC = 56.6 nM). These isolates may represent an extension of a population of parasites from eastern Thailand.