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.     

Declining mefloquine sensitivity of Plasmodium falciparum along the Thai-Myanmar border

Mefloquine sensitivity of Plasmodium falciparum along the Thai-Myanmar border, both in vitro and in vivo, following different first-line treatments for uncomplicated falciparum malaria patients in these areas during the period 1997--2003 were studied. Standard in vitro micro tests and in vivo efficacy according to World Health Organization methodologies were performed. P. falciparum isolates along the Thai-Myanmar border with in vitro sensitivity to mefloquine have had up to a ten-fold decrease in sensitivity compared to a baseline done in 1986, conducted one year after the drug was first introduced to Thailand. The reduction in the mefloquine sensitivity of P. falciparum isolates in Tak Province developed rapidly, with the highest IC50 of 1,254 nM in 1997. The IC50 declined to 1,067 and 737 nM in 1999 and 2001, respectively, but there was no statistically significant difference in the sensitivity. The sensitivity of P. falciparum isolates from Mae Hong Son, Kanchanaburi, and Ranong, where the first line treatment was mefloquine 15 mg/kg single dose, continued to decline, where in 2001 the IC50 were 1,087, 941, and 1,116 nM, respectively, in these provinces. The difference in sensitivities of P. falciparum isolates in Mae Hong Son and Ranong in 2001, compared to 1997, was statistically significant (p<0.05). Good therapeutic efficacy of the artesunate-mefloquine combination in Tak Province was observed. Adequate clinical responses (ACR) were 89.5% and 92.3% in 1997 and 2002, respectively. The efficacy of mefloquine alone in Mae Hong Son, Kanchanaburi, and Ranong has significantly dropped. ACR in 1997 and 2001 in Mae Hong Son were 87.8% and 73.2%, respectively, in Kanchanaburi were 82% and 59.6%, respectively, and in Ranong were 96% and 31.6%, respectively.     

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.     

中缅边境地区恶性疟原虫对氯喹、哌喹、咯萘啶敏感性的体外测定

目的了解中缅边境地区恶性疟原虫（Plasmodium falciparum）对氯喹、哌喹和咯萘啶敏感性的变化。方法 2009年9～12月在中缅边境的缅甸拉咱市采集了51份恶性疟血样,采用Rieckmann体外微量测定法进行药物敏感性测定。结果敏感性测定结果有效的42份血样中、其恶性疟原虫对氯喹、哌喹和咯萘啶的抗性率分别为95.2%、7.1%和54.8%,半数抑制量(ID₅₀)分别为320.5、128.2和96.0 nmol/L。在抗咯萘啶的23份血样中,对氯喹和哌喹的交叉抗性率分别为91.3%（21/23）和13.0%（3/23）;抗氯喹的40份血样中,对哌喹和咯萘啶的交叉抗性率分别为7.5%（3/40）和52.5%（21/40）;抗哌喹的3份血样中,对氯喹和咯萘啶的交叉抗性率均为100%。结论在缅甸拉咱市调查点流行的恶性疟原虫对氯喹已普遍产生抗性,约半数对咯萘啶具有抗性,多数对哌喹则敏感。     

In vitro activity of chloroquine,quinine, mefloquine and halofantrine against Gabonese isolates of Plasmodium falciparum

OBJECTIVES: To determine the in vitro activity of antimalarial drugs against isolates of Plasmodium falciparum in Gabon. METHODS: Plasmodium falciparum isolates were collected from symptomatic infections in the hospitals of Bakoumba and Franceville, south-east Gabon and in 2000. In vitro activity of chloroquine, quinine, mefloquine, halofantrine was measured by the isotopic microtest. RESULTS: A total of 60 and 62 isolates gave interpretable data in Franceville and Bakoumba, respectively. In Franceville, 50.0% (mean IC50 = 111.7 nm), 0% (mean IC50 = 156.7 nm), and 21.2% (mean IC50 = 12.4 nm) of isolates, respectively, showed in vitro resistance to chloroquine, quinine and mefloquine. In Bakoumba, we saw resistance to chloroquine, quinine, mefloquine and halofantrine in 95.0% (mean IC50 = 325.8 nm), 10.2% (mean IC50 = 385.5 nm), 47.5% (mean IC50 = 24.5 nm) and 18.2% (mean IC50 = 1.9 nm) of isolates, respectively. Activities of chloroquine and mefloquine, chloroquine and quinine, and mefloquine and quinine were positively correlated. CONCLUSIONS: Antimalarial drug resistance is high in this area of Gabon. The extent of resistance is disparate, as all tested drugs were less efficacious in Bakoumba than in Franceville.     

Analyses of cytochrome b mutations in Plasmodium falciparum isolates in Thai-Myanmar border

BACKGROUND: The combination of atovaquone and proguanil (Malarone) has been established as a drug of choice to prevent and treat multi-drug resistant Plasmodium (P.) falciparum malaria in travelers. However, several cases of resistance against Malarone have been reported in some parts of Africa, and many of the cases are believed to be associated with mutations at the codon 268 of cytochrome b gene in mitochondria of P. falciparum. The aim of the study was to estimate the effectiveness of Malarone in treatment and prophylaxis for the travelers to Thai-Myanmar border where multi-drug resistant malaria is highly endemic. METHODS: Seventy P. falciparum samples obtained from patients from Thai-Myanmar border were sequenced to detect mutations around the codon 268. The same samples were also sequenced to detect P. falciparum chloroquine resistance transporter mutation (PfCRT K76T). RESULTS: All the 70 samples showed no mutations at the codon 268 of cytochrome b gene. Whereas, 50 samples, whose pfcrt genes were sequenced successfully, had an identical genotype for K76T mutation. CONCLUSION: In Asian countries, even in the multi-drug resistant areas in the great Mekong region, no case of Malarone resistance has been reported clinically or genetically thus far. In this study, all the P. falciparum parasites tested successfully were shown to be chloroquine resistant but atovaquone susceptible genetically. The more the usefulness of Malarone increases for both treatment and prophylaxis, the wider the drug-resistance against Malarone may spread in the region. Although the total number of samples examined is not large, it is concluded from these findings that Malarone should be recommended for prophylaxis of malaria for travelers to the Mekong region.     

In vitro chloroquine susceptibility and PCR analysis of pfcrt and pfmdr1 polymorphisms in Plasmodium falciparum isolates from Senegal

Chloroquine resistance has been linked to mutations in the pfcrt and pfmdr1 genes of Plasmodium falciparum. To estimate the prevalence of the pfcrt K76T, pfmdr1 N86Y, and pfmdr1 D1246Y polymorphisms, isolates of P. falciparum from Senegal, West Africa, were analyzed, and the results were compared to in vitro chloroquine susceptibility. By the in vitro DELI test, 31% of these samples were resistant to chloroquine. Polymerase chain reaction-based assays and confirmatory sequencing demonstrated the pfcrt T76, pfmdr1 Y86, and pfmdr1 Y1246 alleles in 79%, 31%, and 2% of the isolates, respectively. All three mutant alleles were present in both in vitro susceptible and resistant isolates. On the basis of these findings, it appears that these molecular markers are not consistently predictive of in vitro chloroquine resistance in Senegal.     

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.     

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 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.     

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.     

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.     

Molecular analysis of pfatp6 and pfmdr1 polymorphisms and their association with in vitro sensitivity in Plasmodium falciparum isolates from the Thai-Myanmar border

The association between pfatp6, pfmdr1 polymorphisms (gene mutation and amplification) and in vitro susceptibility to mefloquine (MQ), artesunate (AS), quinine (QN), and chloroquine (CQ) was investigated in a total of sixty-three Plasmodium falciparum isolates collected from the Thai-Myanmar border. The mutations of pfatp6 at codons R37K, G639D, S769N, and I898I and of pfmdr1 at codons N86Y, Y184F, N1042D, and D1246Y were analyzed by polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP). Pfatp6 and pfmdr1 gene copy numbers were analyzed by quantitative real time-polymerase chain reaction (qRT-PCR). In vitro susceptibility test was successful in 58 culture-adapted isolates. Median (range) IC(50) values for MQ, AS, QN, and CQ were 28.96 (3.4-100.5), 1.74 (0.8-5.57), 223.9 (14.99-845.47), and 69.93 (9.6-183.18) nM, respectively. There was a significant positive correlation (R(2) = 0.58) of parasite susceptibility to MQ, AS, and QN. Twelve isolates showed marked decline in susceptibility to AS [median (range) IC(50) = 3.78 (3.07-5.57) nM]. Almost all isolates carried wild-type pfatp6 and pfmdr1 alleles at the investigated codons, while only three isolates (5%) carried pfmdr1 mutation alleles at codon 86. Mutation at codon 86 was associated with a significant increase in the susceptibility of parasite isolates to MQ and QN. All of the sixty-three isolates carried only one pfatp6 copy number. Thirty-three out of the 58 isolates showed increase in pfmdr1 gene copies, which was associated with reduced in vitro susceptibility to MQ, AS, and QN. No association between mutation or amplification of pfatp6 gene and in vitro susceptibility of P. falciparum isolates was found.     

Role of Plasmodium falciparum chloroquine resistance transporter and multidrug resistance 1 genes on in vitro chloroquine resistance in isolates of Plasmodium falciparum from Thailand

Resistance to chloroquine is a public health problem worldwide. Polymorphisms of the Plasmodium falciparum chloroquine resistance transporter (pfcrt) and P. falciparum multidrug resistance 1 (pfmdr1) genes have been linked to chloroquine resistance. Although the K76T mutation in the pfcrt gene has been shown to be a key determinant in chloroquine resistance, evidence suggests that the pfmdr1 gene could modulate the level of chloroquine resistance. However, few studies of field isolates could identify the interactive role of these two genes in chloroquine resistance. Thus, we evaluated the influence of pfcrt and pfmdr1 polymorphisms on in vitro chloroquine sensitivity in 89 adapted isolates of P. falciparum from Thailand. We found that 87 of 89 isolates contained the CVIET haplotype of the pfcrt gene. Two additional mutations in the pfcrt gene were identified, i.e., K6Q and H97L. For the pfmdr1 polymorphisms, the 184F allele was common in the parasites isolated along the Thailand-Cambodia border, and those isolated along the Thailand-Myanmar border contained higher copy numbers. Our results indicate that the additional mutations, in particular H97L in the pfcrt gene and Y184F in the pfmdr1 gene and its copy number, influence the level of chloroquine resistance.     

In vitro susceptibility of Plasmodium falciparum isolates from Jilore, Kenya, to antimalarial drugs

Twenty-six Plasmodium falciparum isolates obtained during a prophylaxis study at Jilore primary school, Malindi, Kenya, were adapted to in vitro culture and their susceptibility to 13 antimalarial drugs was tested by a modified radioisotopic method. Pyrimethamine, chloroquine, amodiaquine, cycloguanil, chlorcycloguanil, quinine, quinidine and sulfadoxine, and the experimental compounds MB 35769, mefloquine, WR 184806, parvoquone, and menoctone were used. The isolates could be divided into two groups with significantly different susceptibility to pyrimethamine, shown by a 755-fold difference in the mean ID50 values (2.77 +/- 1.98 x 10(-10) mol/l and 2.09 +/- 1.64 x 10(-7) mol/l). The mean susceptibility of the two groups differed 7.7-fold for chlorcycloguanil and 14.6-fold for cycloguanil, but were not significantly different for the other drugs. All isolates were more sensitive to amodiaquine than to chloroquine in vitro. The ratio of the geometric mean ID50 values of chloroquine to amodiaquine was 3.13. The ratio for the chemically related compounds parvoquone to menoctone was 5.63, quinine to quinidine was 5.58, and mefloquine to WR 184806 was 12.16.     

In vitro and in vivo susceptibility of Plasmodium falciparum isolates from Liberia to pyrimethamine, cycloguanil and chlorcycloguanil

In vivo susceptibility of Plasmodium falciparum to chlorproguanil and in vitro susceptibility to pyrimethamine, cycloguanil and chlorcycloguanil were studied in 38 children from two Liberian villages. Children in one village (Lagbala) had received monthly chemosuppression with chlorproguanil from 1976-1985, and children in the other village (JDF) had received fortnightly chlorproguanil from 1981-1985. The highest and lowest IC100 for pyrimethamine differed by a factor of 10(5), but they differed only by a factor of 10(3) for chlorcycloguanil. The mean IC100 for chlorcycloguanil was significantly lower (P less than 0.0001) than the mean IC100 for pyrimethamine and cycloguanil, and the IC100 for the samples most resistant to chlorcycloguanil (10(-8) M) was still well below peak blood concentrations after chlorproguanil administration. Resistance could be defined as IC100 greater than or equal to 10(-6) M for pyrimethamine and IC100 greater than or equal to 10(-8) M for chlorcycloguanil. The isolates most resistant or most sensitive to pyrimethamine were also the most resistant or most sensitive to chlorcycloguanil, indicating partial cross-resistance between the two drugs. The in vivo response to chlorproguanil 1.5 mg kg-1 in Lagbala was equal to the response in 1983. Chlorproguanil 1.5 mg kg-1 resulted in lower parasite rates on day 3 and 7, but did not prevent 60% of the children requiring treatment with chloroquine during the four weeks' follow-up after chlorproguanil administration.     

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.     

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.