Apichart Nontprasert

Genetic diversity of Plasmodium vivax in Kolkata, India

BackgroundPlasmodium vivax malaria accounts for approximately 60% of malaria cases in Kolkata, India. There has been limited information on the genotypic polymorphism of P. vivax in this malaria endemic area. Three highly polymorphic and single copy genes were selected for a study of genetic diversity in Kolkata strains.MethodsBlood from 151 patients with P. vivax infection diagnosed in Kolkata between April 2003 and September 2004 was genotyped at three polymorphic loci: the P. vivax circumsporozoite protein (pvcs), the merozoite surface protein 1 (pvmsp 1) and the merozoite surface protein 3-alpha (pvmsp 3-alpha).ResultsAnalysis of these three genetic markers revealed that P. vivax populations in Kolkata are highly diverse. A large number of distinguishable alleles were found from three genetic markers: 11 for pvcs, 35 for pvmsp 1 and 37 for pvmsp 3-alpha. These were, in general, randomly distributed amongst the isolates. Among the 151 isolates, 142 unique genotypes were detected the commonest genotype at a frequency of less than 2% (3/151). The overall rate of mixed genotype infections was 10.6%.ConclusionThese results indicate that the P. vivax parasite population is highly diverse in Kolkata, despite the low level of transmission. The genotyping protocols used in this study may be useful for differentiating re-infection from relapse and recrudescence in studies assessing of malarial drug efficacy in vivax malaria.

Therapeutic responses to antibacterial drugs in vivax malaria

Some antibacterial drugs have antimalarial activity that can be exploited for the prevention or treatment of malaria. Monotherapy with tetracycline, doxycycline, clindamycin or azithromycin was assessed in 1995-98 in 92 adult patients in Thailand with Plasmodium vivax malaria. All patients recovered following treatment and the early therapeutic responses were similar among the 4 groups. The overall median fever clearance time was 57 h and the mean (SD) overall time to parasite clearance was 134 (48) h. Of 66 patients who completed a 28-day follow-up, reappearances of vivax infection occurred in 27 patients (41%) from all groups; delayed appearances of falciparum malaria occurred in 6 patients (9%), only from the azithromycin group. The overall mean (SD) time to reappearance of P. vivax was 23 (5) days and time taken for detection of falciparum malaria was 13 (4) days after starting treatment for vivax malaria. The 28-day cumulative cure rates of clindamycin (n = 12), tetracycline (n = 18) and doxycycline (n = 18) groups were similar (P > or = 0.14) and all were significantly higher compared to the azithromycin group (n = 18; P < or = 0.04). The intervals until vivax reappearance were also significantly shorter in the azithromycin group [mean (SD) = 21 (6) vs 25 (3) days, P < 0.05] suggesting that some of these were recrudescences. The apparent success rate (no subsequent appearances of either vivax or falciparum infection) was significantly lower for the azithromycin group (11%) compared to the other groups (34-78%; P < 0.01). In current antibacterial treatment regimens, short-course azithromycin has inferior antimalarial activity compared to clindamycin or the tetracyclines.

Assessment of the neurotoxicity of oral dihydroartemisinin in mice

High doses of the oil-soluble antimalarial artemisinin derivatives artemether and arteether, given by intramuscular injection to experimental mammals, produce an unusual pattern of selective damage to brainstem centres predominantly involved in auditory processing and vestibular reflexes. We have shown recently, in adult Swiss albino mice, that constant exposure either from depot intramuscular injection of oil-based artemisinin derivatives, or constant oral intake carries relatively greater neurotoxic potential than other methods of drug administration. Using the same model, oral dihydroartemisinin suspended in water was administered once or twice daily at different doses ranging from 50 to 300 mg/kg/day for 28 days. The neurotoxic potential of the oral dihydroartemisinin was assessed and compared to that of oral artemether and artesunate. Oral artemether, artesunate, and dihydroartemisinin had similar neurotoxic effects with no significant clinical or neuropathological evidence of toxicity at doses below 200 mg/kg/day. These data indicate that once and twice daily oral administration of artemether, artesunate and dihydroartemisinin is relatively safe when compared to intramuscular administration of the oil-based compounds.

Antimalarial activity and interactions between quinine, dihydroquinine and 3-hydroxyquinine against Plasmodium falciparum in vitro

The antimalarial activities of quinine, dihydroquinine (a natural impurity found in commercial pharmaceutical formulations of quinine) and 3-hydroxyquinine (the principal metabolite of quinine in humans) were tested both individually and in pairs against 5 strains of Plasmodium falciparum isolated from patients in Thailand. The median inhibitory concentrations (IC50) were similar for quinine (168 nmol/L, range 68-366), and dihydroquinine (129 nmol/L, range 54-324), and both were significantly lower than that of 3-hydroxyquinine (1160 nmol/L, range 378-3154) (P = 0.027). When these drugs were tested in combination, there was no evidence of synergy or antagonism, as determined by fractionary inhibitory indices and isobolograms. Quinine and its impurity, dihydroquinine, have equivalent antimalarial activities which are approximately 10 times greater than that of the metabolite 3-hydroxyquinine. These 2 compounds, which are not usually measured in specific drug assays, contribute to antimalarial activity after quinine administration.

Synergy of antithrombin III concentrate and antivenom in preventing coagulopathy in a rat model of Malayan pit viper envenoming

The effects of unrefined equine antivenom and antithrombin III (AT-III) concentrate on the coagulopathy induced by systemic envenomation by Malayan pit viper (Calloselasma rhodostoma; MPV) venom were investigated in a rat model. 37 rats received an intramuscular injection of MPV venom and serial blood samples were taken from the femoral vein for simple whole blood clotting tests and measurement of AT-III activity. 30 min after venom injection, treatment (antivenom, AT-III or both) was given intravenously. 6 rats were untreated and all developed uncoagulable blood and AT-III depletion 90-210 (median 180) min after venom injection. A combination of high dose AT-III concentrate (0.5 units/g) and antivenom (20 micrograms/g) prevented abnormal clotting (P less than 0.001), whereas AT-III alone, antivenom alone, or a combination of low dose AT-III (0.25 units/g) and antivenom did not (P less than 0.05). These results suggest that the coagulation abnormality in MPV envenomation is secondary to activation of the coagulation cascade at several levels, and that treatment with antivenom alone may not be sufficient to reverse or prevent this phenomenon.

Therapeutic effects of antivenom supplemented by antithrombin III in rats experimentally envenomated with Russell's viper (Daboia russelli siamensis) venom

The effects of equine antivenom and antithrombin III (AT-III) on the coagulopathy induced by Russells viper venom (RVV, Daboia russelli siamensis) were investigated in the rat. After taking blood samples from the femoral vein for determination of simple blood clotting time and AT-III activity, all anaesthetized rats received an intramuscular injection of venom (2 micrograms/g). Treatment (antivenom or AT-III or both) was given intravenously through another femoral vein 30 min after venom injection. All untreated rats (n = 7) developed AT-III depletion (< 70%) [mean (S.D.)] 70 (36) min, and incoagulable blood 85 (53) min after venom injection. Supplementation with AT-III (either 0.25 U/g or 0.5 U/g) had no effect on the RVV induced coagulopathy (n = 20). Treatment with antivenom alone (10 micrograms/g) reduced the incidence of abnormal clotting significantly (8/15, 53%) (P = 0.03). When antivenom was given in combination with AT-III (0.5 U/g), abnormal clotting was prevented in all but one animal (1/15, 7%) (P = 0.007). AT-III activity declined progressively in all rats which developed non-clotting blood. These results suggest that coagulopathy in Russells viper envenoming is associated with activation of coagulation and consumption of AT-III. Antivenom can prevent coagulopathy, but its neutralizing activity is augmented significantly by AT-III supplementation.

Antithrombin III and antivenom reversal of coagulopathy in rats envenomated with Malayan pit viper venom

The therapeutic effects of antithrombin III (AT-III) and unrefined equine antivenom in the treatment of coagulopathy induced by Malayan pit viper (Calloselasma rhodostoma) venom were assessed in 42 adult Wistar rats. Following intramuscular venom injection (2 micrograms/g body weight), serial blood samples were taken from the femoral vein for measurement of whole blood clotting time and AT-III activity. There was progressive depletion of AT-III and blood ceased to clot a mean (S.E.) of 164 (8.3) min after venom injection. Coagulopathy was reversed by a high dose antivenom (10 micrograms/g) or a lower dose of antivenom (5 micrograms/g) in combination with AT-III (> or = 0.1 U/g; P < 0.01) but not 5 micrograms/g antivenom or AT-III alone. Following successful treatment, the mean plasma AT-III activity remained above 90%. In this animal model, systemic envenomation by the Malayan pit viper causes uncoagulable blood associated with AT-III consumption. The dose of antivenom required to reverse this coagulopathy can be reduced by half by the addition of AT-III sufficient to maintain blood concentrations within the normal range.