Dennis Kyle

The Shikimate Pathway and Its Branches in Apicomplexan Parasites

The shikimate pathway is essential for production of a plethora of aromatic compounds in plants, bacteria, and fungi. Seven enzymes of the shikimate pathway catalyze sequential conversion of erythrose 4-phosphate and phosphoenol pyruvate to chorismate. Chorismate is then used as a substrate for other pathways that culminate in production of folates, ubiquinone, napthoquinones, and the aromatic amino acids tryptophan, phenylalanine, and tyrosine. The shikimate pathway is absent from animals and present in the apicomplexan parasites Toxoplasma gondii, Plasmodium falciparum, and Cryptosporidium parvum. Inhibition of the pathway by glyphosate is effective in controlling growth of these parasites. These findings emphasize the potential benefits of developing additional effective inhibitors of the shikimate pathway. Such inhibitors may function as broad-spectrum antimicrobial agents that are effective against bacterial and fungal pathogens and apicomplexan parasites.

Development and validation of flow cytometric measurement for parasitemia in cultures of P. falciparum vitally stained with YOYO-1†‡

The need for improved malaria diagnostics has long been recognized.

Mutations in Plasmodium falciparum dihydrofolate reductase and dihydropteroate synthase of isolates from the Amazon Region of Brazil

Since the late 1970s pyrimethamine-sulfadoxine (PS; FansidarTM Hoffman-LaRoche, Basel) has been used as first line therapy for uncomplicated malaria in the Amazon basin. Unfortunately, resistance has developed over the last ten years in many regions of the Amazon and PS is no longer recommended for use in Brazil. In vitro resistance to pyrimethamine and cycloguanil (the active metabolite of proguanil) is caused by specific point mutations in Plasmodium falciparum dihydrofolate reductase (DHFR), and in vitro resistance to sulfadoxine has been associated with mutations in dihydropteroate synthase (DHPS). In association with a proguanil-sulfamethoxazole clinical trial in Brazil, we performed a nested mutation-specific polymerase chain reaction to measure the prevalence of DHFR mutations at codons 50, 51, 59, 108 and 164 and DHPS mutations at codons 436, 437, 540, 581 and 613 at three sites in the Brazilian Amazon. Samples from two isolated towns showed a high degree of homogeneity, with the DHFR Arg-50/Ile-51/Asn-108 and DHPS Gly-437/Glu-540/Gly-581 mutant genotype accounting for all infections in Peixoto de Azevedo (n = 15) and 60% of infections in Apiacás (n = 10), State of Mato Grosso. The remaining infections in Apiacás differed from this predominant genotype only by the addition of the Bolivia repeat at codon 30 and the Leu-164 mutation in DHFR. By contrast, 17 samples from Porto Velho, capital city of the State of Rondônia, with much in- and out-migration, showed a wide variety of DHFR and DHPS genotypes.

Point mutations in the pfcrt and pfmdr-1 genes of Plasmodium falciparum and clinical response to chloroquine, among malaria patients from Nigeria

Abstract Chloroquine (CQ) resistance in Plasmodium falciparum has been associated with specific point mutations in the pfcrt and pfmdr-1 genes. In the present study, 30 children aged 1–12 years, who were all suffering from acute, uncomplicated, P. falciparum malaria in Ibadan, Nigeria, were evaluated to assess the association between these mutations and clinical outcome following treatment with CQ. The parasites, in blood samples collected pre-treatment and, in those who failed treatment, on the day symptoms re-occurred post-treatment, were genotyped using the polymorphic MSP1, MSP2 and GLURP loci and PCR-RFLP. The results showed that, pre-treatment, all 30 patients had polyclonal infections, the mean numbers of P. falciparum clones detected per infection being 2.6 with MSP1, 4.2 with MSP2 and 2.8 with GLURP. The T76 allele of pfcrt and the Y86 allele of pfmdr-1 were found in 53% and 40%, respectively, of the pre-treatment samples from the 15 patients who failed CQ treatment, but the Y1246 mutation in pfmdr-1 was never detected. Although the parasites from the two patients with high-grade (RIII) resistance to CQ had both of these point mutations, the presence of the T76 allele of pfcrt or the Y86 allele of pfmdr-1 (considered individually) could not be used to predict treatment outcome. However, a high frequency of clonal multiplicity may confound attempts to associate the point mutations in pfcrt or pfmdr-1 with clinical response to CQ. It remains unclear whether the present results represent the characteristics of the predominant parasite populations in the study area. Further studies are needed before the strength of the association between the point mutations identified as markers of drug resistance and clinical outcome can be accurately evaluated, in this and other regions of intense transmission.

Reversal of mefloquine resistance with penfluridol in isolates of Plasmodium falciparum from south-west Nigeria

The susceptibilities of isolates of Plasmodium falciparum from Nigeria and two reference cloned strains (D6 and W2) to mefloquine or chloroquine alone and in combination with either penfluridol, a piperidine analogue, or verapamil were determined using a modification of the semiautomated microdilution technique. Six of the isolates showed reduced susceptibility to mefloquine in vitro. The response of the 6 isolates was similar to that of the mefloquine resistant reference clone D6, with 50% inhibitory concentration (IC50) values = 3.29-9.72 ng/ml. Only 2 of the Nigerian isolates were sensitive to mefloquine (IC50 = 1.16 ng/ml and 2.62 ng/ml) and were similar to the reference mefloquine sensitive clone W2 (IC50 = 1.78 ng/ml). All the isolates tested were sensitive to chloroquine, with IC50 values = 1.5-3.04 ng/ml. Simultaneous incubation of the parasites with a constant sub-inhibitory concentration of penfluridol (5.0 x 10(-7)M) and mefloquine increased the susceptibility of the resistant parasites to mefloquine. Addition of the neuroleptic drug penfluridol did not alter the response of sensitive parasites to mefloquine or chloroquine. Similarly, addition of 1.0 x 10(-6)M verapamil did not affect the activity of mefloquine against the sensitive or resistant parasites.

Poor efficacy of antimalarial biguanide-dapsone combinations in the treatment of acute, uncomplicated, falciparum malaria in Thailand

Combinations of dapsone with proguanil or chlorproguanil have proved effective in the treatment of chloroquine-resistant falciparum malaria in Africa and for prophylaxis in Asia. These combinations have not been used for treatment in areas with multi-drug-resistant parasites such as in Thailand. Combinations of dapsone (approximately 4 mg/kg) plus ether proguanil (approximately 8 mg/kg; DP regimen; N = 10) or chlorproguanil (approximately 1.4 mg/kg; DC regimen; N = 16) were given once a day for 3 days to adult Thai patients with acute, uncomplicated, falciparum malaria. The two regimens were well tolerated and had no side-effects, but the cure rates, assessed at 28-day follow-up, were only 10% for DP (60% with RI response and 30% with RII) and 14% for DC (29% with RI response and 57% with RII). The mean (S.D.) fever-clearance times in those patients who were cured (S) or whose infections recrudesced (RI response) were 103 (56) h for those given DP and 90 (42) h for 6 those given DC. The corresponding parasite-clearance times were 83 (46) for DP and 53 (21) h for DC. In-vitro susceptibility testing of isolates obtained both before treatment and at recrudescence demonstrated marked resistance to cycloguanil, dapsone, chloroquine and mefloquine. The results demonstrate that short-course treatment with dapsone plus either proguanil or chlorproguanil is ineffective for the treatment of falciparum malaria in Thailand.

Development and validation of flow cytometric measurement for parasitaemia using autofluorescence and YOYO-1 in rodent malaria.

An automated flow cytometric (FCM) detection method has been developed and validated with a simple diagnostic procedure in parasitized erythrocytes of Plasmodium berghei-infected rats using the nucleic acid-binding fluorescent dye YOYO-1. High levels of reticulocytes were detected during the course of the infection, ranging from 1.2-51.2%, but any RNA potentially confounding the assay could be removed by digestion with RNAse. The cell counts of uninfected, infected, and nucleated cells occurred with high precision. The cells were divided into different populations according to their physical or chemical properties but various factors within the assay such as cell fixation, RNA digestion, and compensation required optimization. In this study, FCM greatly simplified and accelerated parasite detection, with a mean precision of 4.4%, specificity of 98.9% and accuracy of 101.3%. The detection and quantitation limits in the assay were 0.024% and 0.074% parasitaemia, respectively. Overall, the parasite counts by FCM measurement correlated highly (r2=0.954-0.988) with the parasitaemia measured by light microscopical analysis when animals treated with suppressive, clearance, and curative doses of novel antimalarial drugs were examined. The lower levels of parasitaemia (30%) detected by microscopy compared to FCM may be related to a number of schizonts externally attached to the erythrocyte membranes that normally would not be included in microscopy counting. Lower sampling error and reliable identification of rodent erythrocyte parasites based on the principles of FCM have replaced the traditional blood smear in our laboratory.

Efficacy comparison of intravenous artelinate and artesunate in Plasmodium berghei-infected Sprague-Dawley rats.

This paper reports the comparative antimalarial efficacy of intravenous artelinate and artesunate in rats. Prior to efficacy experiments, a Plasmodium berghei-Sprague-Dawley rat model of malaria was developed, in which the clearance effects of intravenous drugs could be readily compared. In efficacy experiments, groups of P. berghei-infected rats were given 3 daily intravenous treatments of artelinate or artesunate at molar equivalent dose rates (total of 0-191.2 micromoles/kg). Artelinate was superior to artesunate in terms of clearance (100% clearance dose of 95.6 micromoles/kg (40 mg/kg) versus 191.2 micromoles/ kg for AS (73.4 mg/kg)) and parasite clearance time (1.7 +/- 0.5 days for AL versus 2.7 +/- 0.5 days for AS at a dose rate of 191.2 micromoles/kg, P < 0.01). No frank clinical toxicity was observed, though both artesunate and artelinate induced dose-related vascular necrosis at the site of injection. The necrosis was less severe and reversible when the drugs were administered via femoral, rather than tail/foot veins. The data suggest that the P. berghei-7-week-old Sprague-Dawley rat model of malaria is reproducible and useful for assessing the efficacy of antimalarials and that artelinate is at least as potent, and safe, as artesunate, the leading clinical treatment for severe malaria.

Structure and antimalarial activity of adducts of 11-azaartemisinin with conjugated terminal acetylenes

Several N-substituted 11-azaartemisinins were prepared from 11-azaartemisinin in high yield by the DMAP catalyzed addition of terminal acetylenes conjugated with electron-withdrawing groups. Their antimalarial activities against two drug-resistant strains of Plasmodium falciparum were determined.

The effects of α1-acid glycoprotein on the reversal of chloroquine resistance in Plasmodium falciparum

Abstract An in-vitro model based on the semi-automated microdilution technique has been developed for selecting compounds that might be used clinically for the reversal of chloroquine resistance. This was used initially to test the susceptibility of Plasmodium falciparum clone W2 to chloroquine (CQ). The model was then employed to investigate the effects of each of four resistance-reversing agents (verapamil, desipramine, chlorpheniramine and promethazine, at 1 μM) on this parasites susceptibility to CQ, with and without α1-acid glycoprotein (AGP), at a patho–physiological concentration (1.25 g/litre), in the culture medium. In the absence of AGP, each of the resistance-reversing agents reduced the median inhibitory concentrations of CQ by 82%–97%, from a baseline value of about 94 ng/ml. In the presence of AGP, however, most of the resistance-reversing agents had much less effect. There appears to be competitive interaction between CQ, the resistance-reversing agents and AGP. The binding kinetics between CQ, resistance-reversing agents, AGP and other plasma proteins will clearly need to elucidated if clinically effective resistance-reversing agents are to be selected in vitro.