In vitro sensitivity of Plasmodium falciparum isolates from Gabon to chloroquine and cycloguanil]

The in vitro susceptibility of 91 Plasmodium falciparum isolates obtained from malaria-infected children living near Libreville (Gabon) was evaluated against chloroquine and cycloguanil (biologically active metabolite of proguanil), using an isotopic micro-drug susceptibility test. In vitro resistance to chloroquine and cycloguanil was observed in 83% (35/42) and in 38% (30/78) of the patients, respectively. Our data showed that 41% (16/39) of Gabonese field isolates were resistant both to chloroquine and cycloguanil. These findings are of great importance because they might indicate imminent chloroquine-proguanil failure, and there are not many affordable antimalarial drugs to replace chloroquine-proguanil combination.     

Patterns of Plasmodium falciparum Drug Resistance in Nonimmune Travellers to Africa

 Falciparum malaria is a major cause of morbidity and mortality among tourists returning from endemic areas. Treatment of infected travellers is frequently standardised, and resistance tests are rarely done as they are difficult to perform and interpret. In vitro tests of resistance to chloroquine, mefloquine, quinine, halofantrine, tetracycline, and sulfadoxine/pyrimethamine were performed on Plasmodium falciparum isolates obtained from 52 German travellers returning from malarious areas in sub-Saharan Africa. All patients were treated successfully with a standard dose of mefloquine. No in vivo resistance was observed. Although 20 (38.5%) isolates showed no signs of in vitro resistance, the remaining 32 (61.5%) were resistant to at least one of the tested antimalarial agents. Of these 32 isolates, 11 were multiresistant. Resistance to chloroquine was most frequently observed (55.8%), followed by sulfadoxine/pyrimethamine (11.5%), mefloquine (9.6%), quinine (3.8%), and halofantrine (1.9%). In vitro resistance to tetracycline was not detected in this study group. Treatment of falciparum malaria without resistance testing appears to be effective in most cases, but possible therapeutic failure due to emerging drug resistance should be kept in mind.     

In-vivo evaluation of Plasmodium falciparum sensitivity to chloroquine in Abidjan

In vivo tests of Plasmodium falciparum chloroquine sensitivity were conducted in October and November, 1988 with 81 children aged 5 to 9 in several districts of Abidjan, Ivory Coast. The WHO standard scheme covering 7 days on basis of 25 mg per kilo spread over 3 days resulted in a therapeutic failure in 29.6 per cent cases. Nevertheless, a drop in overall parasitemia by over 80 per cent was noted from Day-0 to Day-2 in 70 per cent of visible resistance. Only in vivo tests conducted at a later stage with identification of chloroquine in the blood stream, with together in vitro studies will make it possible to know the actual level of resistance of Plasmodium falciparum strains to chloroquine.     

Plasmodium falciparum resistance to cytocidal versus cytostatic effects of chloroquine

With one exception (Gligorijevic et al., Mol Biochem Parasitol 2008;159:7-23.) all previous quantification of chloroquine (CQ) potency vs. P. falciparum has been by growth inhibition assays, meaning potency is defined as cytostatic potential and quantified by IC(50) values. In this study we investigate the cytocidal potency of CQ and other common quinoline antimalarial drugs (quantified as LD(50)). Similar to results from assays for cytostatic potency, we are able to readily distinguish drug resistant from drug sensitive P. falciparum parasites as well as different degrees of resistance. However, we find that fold-resistance to CQ and other quinoline drugs quantified via LD(50) ratios differs quite dramatically from fold resistance calculated via IC(50) ratios. Also, importantly, we find that verapamil chemoreversal of CQ resistance differs when quantified via cytocidal vs. cytostatic assays, as do patterns of "multidrug" resistance in well-known laboratory strains of P. falciparum. The results have important implications for development of new antimalarial drugs and for fully defining the genetic loci that confer clinically relevant antimalarial drug resistance phenomena.     

A fluorometric sensitivity test for chloroquine in Plasmodium falciparum isolates from patients

A new technique for assessment of in vitro growth of Plasmodium falciparum by fluoroassay was used to determine the sensitivity to chloroquine of parasites from cases of malaria imported into Japan. The technique was reliable, giving comparable results to the Rieckmann test, and is also applicable in the field.     

In vitro sensitivity pattern of chloroquine and artemisinin in Plasmodium falciparum

Artemisinin (ART) and its derivatives form the mainstay of antimalarial therapy. Emergence of resistance to them poses a potential threat to future malaria control and elimination on a global level. It is important to know the mechanism of action of drug and development of drug resistance. We put forwards probable correlation between the mode of action of chloroquine (CQ) and ART. Modified trophozoite maturation inhibition assay, WHO Mark III assay and molecular marker study for CQ resistance at K76T codon in Plasmodium falciparum CQ-resistant transporter gene were carried out on cultured P. falciparum. On comparing trophozoite and schizont growth for both CQ-sensitive (MRC-2) and CQ-resistant (RKL-9) culture isolates, it was observed that the clearance of trophozoites and schizonts was similar with both drugs. The experiment supports that CQ interferes with heme detoxification pathway in food vacuoles of parasite, and this may be correlated as one of the plausible mechanisms of ART.     

Plasmodium falciparum drug resistance and sulfadoxine-pyrimethamine in Africa

The sulfadoxine-pyrimethamine combination has not been recommended for the prophylaxis of malaria since 1985 following serious accidents in the USA. However, this drug is worth considering for treatment since it has the advantage over mefloquine of being cheaper, having fewer side effects and it avoids using mefloquine. A study of Plasmodium falciparum resistance to Fansidar should be carried out on cases imported to France to determine an adapted utilisation of this drug. This would be an appreciable advantage for tropical Africa.     

DNA hybridization for assessment of response of Plasmodium falciparum to chloroquine therapy.

We studied the accuracy of PFR1-AP, a synthetic DNA hybridization probe conjugated to alkaline phosphatase, in monitoring Plasmodium falciparum parasitemia during in vivo drug susceptibility surveys. Duplicate blood samples were collected from six children enrolled in a 14-day in vivo chloroquine study in Rwanda. Results obtained by microscopic examination of Giemsa-stained thick blood smears and by DNA hybridization were compared. Both techniques successfully monitored an infection with chloroquine-susceptible parasites and infections exhibiting various levels of resistance to treatment. For each patient, temporal evolution of the microscopic parasite counts and the DNA hybridization signals were closely parallel, although a wide range of rapidly changing levels of parasitemia occurred through the course of the study. This suggests that DNA hybridization assay using PFR1-AP detects P. falciparum parasites sensitively and specifically and is a valuable tool for drug resistance surveys.     

Analysis of pfcrt point mutations and chloroquine susceptibility in isolates of Plasmodium falciparum

Recent transfection based studies demonstrated that cg2, a candidate gene for chloroquine resistance in Plasmodium falciparum, was not the resistance determinant. A further analysis of the initial 36 kb locus comprising the cg2 gene led to the discovery of another gene, pfcrt, which was absolutely associated with chloroquine resistance in forty parasite lines [Fidock DA, Nomura T, Talley AT, Su XZ, Cooper R, Dzekunov SM, Ferdig MT, Ursos LMB, Sidhu ABS, Naudé B, Deitsch KW, Su XZ, Wootton JC, Roepe PD, Wellems TE. Mutations in the P. falciparum digestive vacuole transmembrane protein PfCRT and evidence for their role in chloroquine resistance. Mol Cell 2000;6:861-71]. The aim of this study was to evaluate, in 146 unselected clinical isolates obtained mostly from non-immune travellers returning from various endemic countries to France in years 1995-1999, the association between in vitro chloroquine resistance and the sequence of a part of the pfcrt gene. For comparison, the determination of the cg2 kappa and the pfmdr1 codon 86 genotypes were also performed on the same isolates. As determined by an isotopic semi-microtest, 70 isolates were susceptible to chloroquine (50% inhibitory concentration<80 nM) and 76 were resistant. The amplification of a portion of the pfcrt gene spanning codons 72-76, followed by sequencing showed three distinct genotypes: one type associated with susceptible isolates, one type associated mostly with resistant isolates and one type found in a resistant isolate originating from South America. Three different zones could be defined according to the status of codon 76. For 50% inhibitory concentration values< or =40 nM (n=47), all isolates but one had K76 (wild type). For 50% inhibitory concentration values located between 40 and 60 nM, isolates had either K76 (n=5) or K76T (mutant type) (n=6). For 50% inhibitory concentration values>60 nM (n=88), all isolates had K76T. A lack of a strong association between the pfmdr1 N86Y mutation and in vitro chloroquine resistance was observed. Cg2 genotypes were less strongly linked than pfcrt genotypes with in vitro chloroquine susceptibility in isolates located below 40 and above 60 nM. Further studies are needed to determine the reliability of the pfcrt gene as a genetic marker for chloroquine resistance.     

Chemoresistance of Plasmodium falciparum in Kinshasha. In vivo tests and chloroquine absorption

With the test in vivo and the bleeding dosage of chloroquine, authors report the rate of resistance of chloroquine to Plasmodium falciparum. 2.3% of Plasmodium falciparum in Kinshasa are resistant to chloroquine.     

In vitro sensitivity of Plasmodium falciparum to chloroquine in the highland region of Madagascar in 1987

With the Le Bras's isotopic semi-microtest method, the authors have studied 139 strains of Plasmodium falciparum isolated in a village near Tananarive in the Highlands of Madagascar. Conditional malariometric rates show the increase of the recrudescence of Malaria in the area which was recently considered as "surveillance area". 5.8% of the 139 studied strains show an in vitro resistance higher than 120 nmoles/l, but whose the level is ever lower than in Africa. 4.3% exhibit a level between 90 and 120 nmoles/l. These values give better prospect for the Isle because the resistance strain rates have remain nearly stable since 1982. The resistance level is always low.     

Analysis of kappa and omega repeats of the cg2 gene and chloroquine susceptibility in isolates of Plasmodium falciparum from sub-Saharan Africa.

The correlation between the structure of two short sequences from the Plasmodium falciparum cg2 gene and parasite chloroquine susceptibility was evaluated in unselected clinical isolates obtained from travellers returning mainly from Africa to France in 1995 and 1996. As determined by an isotopic semi-microtest, 74 isolates were susceptible to chloroquine (50% inhibitory concentration < 80 nM), 13 were intermediate (80 nM < 50% inhibitory concentration < 110 nM) and 53 were resistant (50% inhibitory concentration > 110 nM). Two polymerase chain reaction assays were developed, one for the kappa and one for the omega repeat domains of cg2 gene. The kappa and the omega repeat domains of 99 isolates were sequenced. A variation in the unit number of kappa and omega repeats was observed. Variations in repetitive sequences, which were not previously described, were found: three for the kappa repeat region: kappa9; kappa10 and kappa11 and three for the omega repeat region: omega8; omega9 and omega22. A polymorphism was observed inside the repeat units of kappa and omega regions. There were six possible kappa repeat units and seven possible omega repeat units. The presence of a particular pattern, containing kappa14 and omega16 repeat units, was associated with a lack of chloroquine susceptibility in 44 out of 46 cases. However, not all resistant isolates had this 'resistant' genotype. Among 43 resistant isolates, 36 (84%) had the kappa14 repeats sequence and 36 had the omega16 repeats sequence. These results lend further support to linkage between cg2 polymorphisms and chloroquine resistance without excluding the existence of other resistance component(s).     

In vivo and in vitro analysis of chloroquine resistance in Plasmodium falciparum isolates from Senegal

To determine the predictive value of chloroquine (CQ) resistance markers in Senegal, Plasmodium falciparum DNA polymorphisms in pfmdr1and pfcrt were examined in relation to clinical outcome. Despite CQ treatment, 17% of patients had parasitemia after 28 days. Examination of molecular markers of CQ resistance revealed that 64% of all isolates had the T76 resistant allele at the pfcrt locus, while 30% carried the Y86 resistant allele at the pfmdr1 locus. The pfcrt T76 allele was present not only in all in vivo resistant isolates, 89% of in vitro resistant isolates, but also in 35% of in vitro sensitive isolates. The pfmdr1 N86Y polymorphism did not correlate with in vitro or in vivo CQ resistance. Our data suggest that the pfcrt T76 allele alone is required but not a sufficient predictor for in vivo CQ resistance.