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.     

In vitro antimalarial susceptibility profile and prcrt/pfmdr-1 genotypes of Plasmodium falciparum field isolates from Malawi

We measured in vitro antimalarial drug susceptibility of 84 Plasmodium falciparum field isolates from Blantyre, Southern Malawi, using the WHO microtest and the lactate dehydrogenase assay. We also genotyped these isolates to investigate whether variation in their absolute drug sensitivity is associated with specific sets of pfcrt and pfmdr-1 mutations harbored by parasites. Our results show that nearly a decade after the withdrawal of chloroquine (CQ) as a first-line antimalarial in Malawi, most isolates are now sensitive to CQ and none is CQ-resistant as predicted by their drug sensitivity phenotype and pfcrt genotype. We also found that these isolates are uniformly sensitive to a range of quinoline-based antimalarials and artemisinin derivatives. These findings reinforce previous reports about a reduction in the proportion of CQ-resistant parasites after the withdrawal of CQ in 1993 and pave the way for reassessing the clinical usefulness of CQ, artemisinins and other quinoline-based antimalarials in Malawi.     

The changing in vitro susceptibility pattern to pyrimethamine/sulfadoxine in Plasmodium falciparum field isolates from Kilifi, Kenya

Two clinical trials that used Falcidin (Cosmos Ltd., Nairobi, Kenya), the antifolate combination of pyrimethamine/sulfadoxine (PM/SD), as treatment for non-severe falciparum malaria in children at Kilifi, Kenya in 1987-1988 and 1993-1995 have presented an opportunity to assess in vitro the susceptibility trend of Plasmodium falciparum to PM and SD over time on the Kenya coast. The first set of isolates was collected prior to the introduction of PM/SD into the Kenya Medical Research Institute/Wellcome Trust Research unit while the second set was taken soon after PM/SD was introduced in the study area as the first-line treatment drug for uncomplicated falciparum malaria. In the first trial, 69 isolates collected before and after treatment of malaria with PM/SD were tested directly in the field for susceptibility to PM and SD using the standard in vitro micro-test technique, with minimal levels of folate. In the second trial, 97 isolates similarly collected were adapted to culture, and tested as described elsewhere. In both studies, PM and SD susceptibility tests were done separately. There was a highly significant decrease (P < 0.01) in the in vitro sensitivity of P. falciparum isolates to PM and SD between the two trials. In the first trial, the isolates were either sensitive to both PM and SD or resistant to PM and sensitive to SD. During the second trial, isolates were either resistant to PM and sensitive to SD or resistant to both drugs. These results are important in estimating the useful therapeutic life (UTL) of PM/SD in this area and in identifying alternative antifolate drugs.     

In vitro antimalarial drug susceptibility and pfcrt mutation among fresh Plasmodium falciparum isolates from the Lao PDR (Laos)

Recent drug trials in Laos have shown high levels of Plasmodium falciparum resistance to chloroquine, but there are no published data on in vitro antimalarial drug susceptibility. We used the double-site enzyme-linked pLDH immunodetection (DELI) assay to estimate the in vitro antimalarial drug susceptibility of 108 fresh P. falciparum isolates from southern Laos. The geometric mean (95% confidence interval) 50% inhibitory concentration values (nmol/L) were 152.4 (123.8-187.6) for chloroquine, 679.8 (533.8-863.0) for quinine, 45.9 (37.9-55.7) for mefloquine, 5.0 (4.4-6.4) for artesunate, 6.3 (4.5-8.9) for dihydroartemisinin, and 59.1 (46.4-75.3) for lumefantrine. The proportion of isolates defined as resistant were 65%, 40%, and 8% for chloroquine, quinine, and mefloquine, respectively. Of 53 isolates genotyped for the pfcrt T76K chloroquine-resistance mutation, 48 (91%) were mutants. P. falciparum in Laos is multi-drug resistant; antimalarial immunity resulting from the use of ineffective chloroquine before 2005 probably contributes significantly to the therapeutic responses in clinical trials.     

In vitro sensitivity of multiresistant Plasmodium falciparum to new candidate antimalarial drugs in western Thailand

The present study, carried out in 1987 in Thailand, has been designed to validate the in vitro microtest system, standardized by the World Health Organization (WHO), for the new antimalarials pyronaridine and halofantrine. The sensitivity of naturally acquired, multiresistant populations of Plasmodium falciparum has been assessed in order to develop a data base for further longitudinal investigations. For both drugs the in vitro microtest system seems to be suitable. The concentration range of plates can be considered as almost ideal for pyronaridine (0.1-6.40 mumol/l) while for halofantrine (0.002-0.128 mumol/l) an upward extension of the concentration range would be appropriate. Validation studies with artemisinin demonstrated the need for revising the protocol for the production of the dosing solutions. In the light of current knowledge about therapeutic concentration levels it would probably be appropriate to adopt a range of 0.2-12.8 mumol/l. All tested isolates, except possibly three, showed sensitive responses to pyronaridine. The high EC99 value of halofantrine could be indicative of some resistance to this drug. Rank correlation analysis suggested cross-resistance of pyronaridine and chloroquine which could be of consequence for the future introduction of pyronaridine.     

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 sensitivity of Plasmodium falciparum to conventional and novel antimalarial drugs in Papua New Guinea

Objective Recent clinical studies have shown high rates of malaria treatment failure in endemic areas of Papua New Guinea (PNG), necessitating a change of treatment from chloroquine (CQ) or amodiaquine (AQ) plus sulphadoxine‐pyrimethamine to the artemisinin combination therapy (ACT) artemether plus lumefantrine (LM). To facilitate the monitoring of antimalarial drug resistance in this setting, we assessed the in vitro sensitivity of Plasmodium falciparum isolates from Madang Province. Methods A validated colorimetric lactate dehydrogenase assay was used to assess growth inhibition of 64 P. falciparum isolates in the presence of nine conventional or novel antimalarial drugs [CQ, AQ, monodesethyl‐amodiaquine (DAQ), piperaquine (PQ), naphthoquine (NQ), mefloquine (MQ), LM, dihydroartemisinin and azithromycin (AZ)]. Results The geometric mean (95% confidence interval) concentration required to inhibit parasite growth by 50% (IC₅₀) was 167 (141–197) nm for CQ, and 82% of strains were resistant (threshold 100 nm ), consistent with near‐fixation of the CQ resistance‐associated pfcrt allele in PNG. Except for AZ [8.351 (5.418–12.871) nm ], the geometric mean IC₅₀ for the other drugs was <20 nm . There were strong associations between the IC₅₀s of 4‐aminoquinoline (CQ, AQ, DAQ and NQ), bisquinoline (PQ) and aryl aminoalcohol (MQ) compounds suggesting cross‐resistance, but LM IC₅₀ only correlated with that of MQ. Conclusions Most PNG isolates are resistant to CQ in vitro but not to other ACT partner drugs. The non‐isotopic semi‐automated high‐throughput nature of the Plasmodium lactate dehydrogenase assay facilitates the convenient serial assessment of local parasite sensitivity, so that emerging resistance can be identified with relative confidence at an early stage.     

In vitro activities of ferrochloroquine against 55 Senegalese isolates of Plasmodium falciparum in comparison with those of standard antimalarial drugs

The in vitro activities of ferrochloroquine, chloroquine, quinine, mefloquine, halofantrine, amodiaquine, artesunate, atovaquone, cycloguanil and pyrimethamine were evaluated against Plasmodium falciparum isolates from Senegal (Dielmo, Ndiop), using an isotopic micro-drug susceptibility test. The IC50 values for ferrochloroquine ranged from 0.55 to 28.2 nM and the geometric mean IC50 for the 55 isolates was 7.9 nM (95% CI, 6.5-9.7 nM). Ferrochloroquine was 35 times more active than chloroquine (35-fold greater against chloroquine-resistant isolates), quinine, mefloquine, amodiaquine, cycloguanil and pyrimethamine. Weak positive correlations were observed between the responses to ferrochloroquine and that to chloroquine, quinine, and amodiaquine, but not compulsorily predictive of cross-resistance. There was no significant correlation between the response to ferrochloroquine and that to mefloquine, halofantrine, artesunate, atovaquone, cycloguanil and pyrimethamine. Ferrochloroquine may be an important alternative drug for the treatment of chloroquine-resistant malaria.     

In vitro activity of halofantrine and its relationship to other standard antimalarial drugs against African isolates and clones of Plasmodium falciparum.

The in vitro activity of halofantrine was studied in chloroquine-susceptible and chloroquine-resistant African clones of Plasmodium falciparum over a period of six months. The susceptibility level remained stable in both clones. The chloroquine-susceptible clone (50% inhibitory concentration [IC50] 6.88 nM) was less susceptible to halofantrine than the chloroquine-resistant clone (IC50 2.98 nM). Using an isotopic semimicro drug susceptibility test, the in vitro activity of halofantrine was compared with the activities of chloroquine, quinine, and mefloquine to study the cross-resistance patterns against 76 African isolates of P. falciparum isolated from cases of malaria imported into France. Chloroquine-resistant isolates (n = 47) were significantly less susceptible to quinine (IC50 234 nM), but were more susceptible to both mefloquine (IC50 3.20 nM) and halofantrine (IC50 1.14 nM) compared with the chloroquine-susceptible isolates (n = 29; IC50 147 nM for quinine, 7.16 nM for mefloquine, and 2.62 nM for halofantrine). A significant positive correlation was found between the activities of chloroquine and quinine and between those of mefloquine and halofantrine, indicating cross-resistance between these drugs, while a negative correlation was observed between chloroquine and mefloquine or halofantrine. The responses to quinine and mefloquine or halofantrine showed no correlation with each other. These results reinforce the importance of a cautious use of antimalarial drugs in Africa.     

Sensitivity of Plasmodium falciparum to antimalarial drugs in Colombia

Sensitivity of Plasmodium falciparum to several antimalarial drugs was determined by in vitro and in vivo tests. Chloroquine resistance in vitro was detected in 97 of 101 patients from different geographic areas of Colombia. Sensitivity to amodiaquine in vitro was observed in 29 of 30 P. falciparum isolates. In vitro sensitivity to amodiaquine was observed in 16 patients infected with chloroquine-resistant P. falciparum. In vitro sensitivity to quinine was demonstrated in 57 P. falciparum isolates. Two infections from the Amazon base (2/24) were resistant to mefloquine in vitro at concentrations of 5.7 and 16 pmol/well. Resistance to Fansidar, a sulfadoxine-pyrimethamine combination, was described in 9 patients from the Amazon region. One patient showed recrudescence of the infection 41 days after treatment. The current distribution and degree of resistance of P. falciparum to widely used antimalarial drugs requires the evaluation of therapeutic schemes based on combinations of fast blood schizontocides with slow acting drugs. These associations may reduce the development of multidrug-resistant isolates and retard the spread of resistant populations of P. falciparum parasites.     

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.     

In vitro susceptibility and genetic variations for chloroquine and mefloquine in Plasmodium falciparum isolates from Thai-Myanmar border

In vitro drug susceptibility to chloroquine (CQ) and mefloquine (MF) were assessed in 39 P. falciparum isolates from the Thai-Myanmar border area. To further characterize CQ- and MF-resistance profiles in this area, we also analyzed pfcrt K76T mutation that is critical for CQ resistance, and pfmdr1 polymorphism that has an association with MF resistance. Eighteen isolates were successfully examined by in vitro tests for CQ, and 17 of them had resistance to the drug. Geometric mean concentration of CQ that inhibited the growth of parasites at 50% (IC50) was 371 +/- 227 nM (105-971 nM). Sixteen isolates were successfully examined by in vitro tests for MF, and 8 of them were resistant to the drug. Geometric mean of IC50 for MF was 41 +/- 31 nM (4-125 nM). Genotypes of drug resistance, such as pfcrt and pfmdr1 mutations, were also analyzed. All the 39 isolates had the same haplotype (CVIET) for PfCRT at its 72-76th amino acids. A pfmdr1 Y86 mutation was found in 95% of isolates. A pfmdr1 D1042 mutation was also present in 7 isolates, while no pfmdr1 Y1246 mutation was observed. These results indicated a correlation between CQ resistance and the pfcrt T76 and pfmdr1 Y86 mutations.     

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.     

The in-vitro susceptibilities of Ghanaian Plasmodium falciparum to antimalarial drugs

In Ghana in 2004 (when choroquine was still the nationally recommended drug for the first-line treatment of malaria), the sensitivities, to chloroquine, amodiaquine, quinine, mefloquine, artesunate and halofantrine, of 60 Plasmodium falciparum isolates from two ecologically distinct areas of the country were assessed in vitro. The aim was to make available, to policy-makers, the field-based evidence needed to review the national strategy for malaria treatment. Drug susceptibilities were explored using the standardized protocol of the Antimalarial Drug Resistance Network. Although 32 of the P. falciparum isolates evaluated (56.1% of the 57 isolates successfully investigated for their susceptibility to choroquine) showed resistance to chloroquine and two showed slightly reduced sensitivity to amodiaquine, all the isolates were sensitive to mefloquine, artesunate, quinine and halofantrine. The median inhibitory concentrations (IC(50)) of chloroquine were positively correlated with those of quinine (r=0.4528; P=0.0008) but not those of any of the other drugs investigated. The IC(50) of amodiaquine and artesunate were also positively correlated (r=0.3703; P=0.0067). These results provide evidence of the presence, in Ghana, of P. falciparum isolates that are highly resistant to chloroquine but generally sensitive to most of the other antimalarial drugs commonly used in the country. Partly in consequence of these observations, the recommended first-line treatment for malaria in Ghana was changed to an amodiaquine-artesunate combination in January 2005.     

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。     

Molecular epidemiology of Plasmodium falciparum resistance to antimalarial drugs in Indonesia

The extent of gene polymorphisms associated with resistance to chloroquine and sulfadoxine-pyrimethamine was examined in field isolates of Plasmodium falciparum from Indonesia. Eight malaria-endemic areas, representing a broad region of the western and eastern Indonesian Archipelago were surveyed. Blood from 20-50 patients was collected at each site, DNA was isolated, and the sequences of four different genes (dihydrofolate reductase [dhfr], dihydropteroate synthase [dhps], P. falciparum multidrug resistance 1 [pfmdr1], and P. falciparum chloroquine resistance transporter [pfcrt]) were analyzed using polymerase chain reaction and restriction fragment length polymorphisms to detect polymorphisms previously shown to be associated with resistance. This analysis identified polymorphisms in dhfr at 108-Asn/Thr, 16-Val, and 59-Arg. Polymorphisms in dhps were found less frequently, either 437-Gly alone or paired with 540-Glu. The pfcrt 76-Thr polymorphism was fixed in all parasite populations and pfmdr1 86-Tyr polymorphisms in all populations except in the most eastern regions. The pfmdr1 1042-Asp polymorphism occurred less frequently. These findings indicate that polymorphisms in genes associated with drug resistance in P. falciparum are found across a broad region of Indonesia.     

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.     

我国恶性疟抗药性的研究现状

本文概述了近年国内恶性疟原虫抗药性的研究进展，包括抗药性的检测方法、抗药性的产生机制以及解决恶性疟抗药性途径的探索。     

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.