Antimalarial activity of tigecycline, a novel glycylcycline antibiotic.

Tigecycline is a novel glycylcycline antibiotic with a broad antibacterial spectrum. Tigecycline was tested with 66 clinical isolates of Plasmodium falciparum from Bangladesh using the histidine-rich protein 2 in vitro drug susceptibility assay. The 50% and 90% inhibitory concentrations of tigecycline were 699 (95% confidence interval, 496 to 986) and 5,905 nM (4,344 to 8,028). Tigecycline shows no activity correlation with traditional antimalarials and has substantial antimalarial activity on its own.

The dynamics of drug resistance in Plasmodium falciparum.

Drug resistance of Plasmodium falciparum is not a recent phenomenon, but it became a major problem when the parasite became resistant to chloroquine, the cheapest and initially the most effective antimalarial compound that could be used for treatment and suppression. In some areas this problem is compounded by resistance to the first line of alternative drugs, and rapid loss of sensitivity to the next line. The dynamics of drug resistance are regulated mainly by drug related selection pressure and intensity of malaria transmission. Mass drug administration in its various forms, and insufficient treatment are obviously the most important motors of selection.

Pharmacodynamic interactions of amodiaquine and its major metabolite desethylamodiaquine with artemisinin, quinine and atovaquone in Plasmodium falciparum in vitro.

The antimalarial in vitro activities of amodiaquine and desethylamodiaquine in combination with atovaquone, quinine and artemisinin against Plasmodium falciparum were investigated in strains F-32, FCR-3 and K-1. These parasitic strains have different sensitivity profiles to the standard antimalarial chloroquine, but all can be considered sensitive to the test drugs, representing the recommended situation for introduction of two partner drugs in combination therapy. Amodiaquine showed marked synergism when combined with each of the three partner compounds at concentration ratios between 90 and 9x10(-7), including the therapeutically relevant range. The interaction profiles of desethylamodiaquine with quinine and artemisinin also showed predominantly synergism over a wide range of concentration ratios between 70 and 9x10(-7). The responses to all combinations exhibited signs of strain-specificity, but such phenomena were usually observed outside the therapeutic range of the concentration ratios. Synergism was generally more marked with increasing EC values, i.e. at concentrations expected to be therapeutically effective and thus clinically relevant. Even trace quantities of amodiaquine were able to potentiate the activity of structurally unrelated antimalarial drugs.

A new and simple solid-phase extraction method for LC determination of pyronaridine in human plasma.

A new approach using a simple solid-phase extraction technique has been developed for the determination of pyronaridine (PND), an antimalarial drug, in human plasma. After extraction with C18 solid-phase sorbent, PND was analyzed using a reverse phase chromatographic method with fluorescence detection (at lambda(ex)=267 nm and lambda(em)=443 nm). The mean extraction recovery for PND was 95.2%. The coefficient of variation for intra-assay precision, inter-assay precision and accuracy was less than 10%. The quantification limit with fluorescence detection was 0.010 microg/mL plasma. The method described herein has several advantages over other published methods since it is easy to perform and rapid. It also permits reducing both, solvent use and sample preparation time. The method has been used successfully to assay plasma samples from clinical pharmacokinetic studies.

Synergism of benflumetol and artemether in Plasmodium falciparum.

Using a continuous in vitro culture system, the sensitivity of Plasmodium falciparum to artemether and a new antimalarial drug, benflumetol (lumefantrine), alone and in combination, was investigated with a multiresistant strain (T-996) from Thailand and a chloroquine-resistant strain (LS-21) from India. Both strains showed similar 50% inhibitory concentration (IC50) levels with artemether alone or benflumetol alone, but the IC90 was higher in strain T-996 compared with strain LS-21: for artemether, 34.45 and 7.11 nmol/L (10.28 and 2.12 ng/ml of erythrocyte-medium mixture [EMM]), and for benflumetol, 293.03 and 95.61 nmol/L (154.69 and 50.47 ng/ml of EMM). When tested in association at artemether:benflumetol (mol: mol) ratios between 100:1 and 1:100, substantial synergism was seen in both strains, especially at the IC90 and IC99 levels. This phenomenon resembles the synergistic interaction of artemisinin derivatives and mefloquine, first observed in laboratory models and later confirmed in clinical experience.

Emergence of multidrug-resistant Plasmodium falciparum in Thailand: in vitro tracking.

Mefloquine was introduced into Thailand in 1985 for the treatment of Plasmodium falciparum infection. Recently, clinical failure of mefloquine was observed in southeastern Thailand, where an epidemic of falciparum malaria occurred. Beginning in 1984 and continuing until 1989, in vitro monitoring of P. falciparum isolates from Borai, a border district in the southeastern part of the country, showed a progressive decrease in mefloquine sensitivity until 1989; in 1990, the degree and prevalence of resistance accelerated. A similar pattern of resistance was observed for halofantrine, an antimalarial drug not yet commercially available in Thailand. In vitro sensitivity patterns of mefloquine and halofantrine elsewhere in the country remained relatively unchanged. These observations suggest a serious deterioration in available drugs for the treatment of falciparum malaria in southeastern Thailand that is predicted to spread throughout the country and Southeast Asia.

Clinical study of pyronaridine for the treatment of acute uncomplicated falciparum malaria in Thailand.

One hundred one adult patients with acute uncomplicated falciparum malaria were treated with pyronaridine. All patients were admitted to the Bangkok Hospital for Tropical Diseases for 28 days to exclude reinfection. Sixty-nine patients (Group I) received pyronaridine 1,200 mg over a three-day period and 32 patients (Group II) received 1,800 mg pyronaridine over a five-day period. Cure rates for the two groups were 63% (38 of 60) for Group I and 88% (23 of 26) for Group II (P<0.05). No RII or RIII type response was seen. Mean fever and parasite clearance times were not significantly different in the two groups. The drug was well-tolerated. In vitro drug sensitivity tests of the paired parasite isolates obtained prior to treatment and after recrudescence indicated that the Plasmodium falciparum isolates of the successfully treated patients had a lower mean concentration for 50% inhibition of growth (IC50) and a much narrower range of the individual IC50 values (15.69 +or- 3.82 ng per ml (mean +or- SD)) as compared with those from the recrudescence cases (22.98 +or- 12.05 ng per ml). Nevertheless, there was no evidence of an increase of the IC50 and IC95 values after recrudescence. The results of the study show that pyronaridine alone at a total dose of 1,800 mg given over five days is well-tolerated in patients suffering from acute uncomplicated malaria and has evident activity against multidrug-resistant falciparum malaria. However, it cannot be recommended for use in Thailand as long as the recrudescence rate is as high as 12%. Further studies of its combinations with other antimalarial drugs are needed.

In vivo-in vitro model for the assessment of clinically relevant antimalarial cross-resistance.

Cross-resistance may be considered one of the most important factors leading to decreased drug susceptibility of Plasmodium falciparum. The study aimed to determine whether clinically relevant cross-sensitivity of P. falciparum existed between artemisinin and mefloquine. Seventy-six patients with falciparum malaria were admitted and treated with artemisinin derivatives. Treatment response parameters were assessed and in vitro drug sensitivity tests were performed with artemisinin, mefloquine, quinine, and chloroquine. Distinct in vitro cross-sensitivity between artemisinin and mefloquine was observed (p = 0.604; P < 0.001). To assess the relevance of this finding for clinical cross-resistance, we used an analytical model based on the relation of in vivo treatment response parameters (fever, parasite and symptom clearance) to a single reference drug with in vitro drug sensitivity data of several other drugs. Artemisinin (R = 0.554; P = 0.009) and mefloquine (R = 0.615; P = 0.002) in vitro drug sensitivities were equally well reflected in the in vivo treatment response to artemisinin, thereby suggesting the clinical relevance of in vitro cross-sensitivity.

Pentoxifylline as an ancillary treatment for severe falciparum malaria in Thailand.

Pentoxifylline, an inhibitor of tumor necrosis factor, has been evaluated as an antimalarial agent in combination with artesunate in 45 patients with severe falciparum malaria. Patients were admitted to the intensive care unit at the Hospital for Tropical Diseases in Bangkok, Thailand, and randomly assigned to treatment for 72 hr with a combination of intravenously administered artesunate and 1) placebo, 2) low-dose pentoxifylline (0.83 mg/kg/hr), or 3) high-dose pentoxifylline (1.67 mg/kg/hr). All 45 patients had one or more manifestations of severe malaria such as cerebral malaria (n = 18), renal failure requiring hemodialysis (n = 9), azotemia (n = 8), jaundice (n = 25), or hyperparasitemia (n = 30). The overall severity was comparable in the three groups. Clinical outcome was assessed with respect to the parasite clearance time and the fever clearance time in all patients. In addition, a number of subsidiary outcome variables were examined in specific subgroups, including the recovery time from coma for patients with cerebral malaria, the duration of intubation in patients with respiratory distress, the number of hemodialysis treatments needed for patients with acute renal failure, and the number of units of blood administered to patients requiring transfusion. Concentrations of tumor necrosis factor were reduced in all three groups at 48 hr after treatment. No significant differences among the three treatment groups were found for any of the outcome variables examined. We conclude that the addition of pentoxifylline to artesunate therapy for severe malaria produced no evident clinical benefit.

PIP: Pentoxifylline, an inhibitor of tumor necrosis factor, was evaluated as an antimalarial agent in combination with artesunate in 45 patients with severe falciparum malaria admitted to the Bangkok (Thailand) Hospital for Tropical Diseases, in a 5-month period in 1994. All patients had 1 or more clinical manifestations of severe malaria, including cerebral malaria (n = 18), renal failure requiring dialysis (n = 9), azotemia (n = 8), jaundice (n = 25), or hyperparasitemia (n = 30). Patients were randomly assigned to receive treatment for 72 hours with a combination of intravenously administered artesunate and either placebo (n = 15), low-dose (0.83 mg/kg/hour) pentoxifylline (n = 15), or high-dose (1.67 mg/kg/hour) pentoxifylline (n = 15). Overall severity was comparable in all 3 groups. Concentrations of tumor necrosis factor were reduced in all 3 groups 48 hours after treatment. There were no significant differences between groups in terms of parasite and fever clearance time, recovery time from coma in patients with cerebral malaria, duration of intubation in patients with respiratory distress, number of hemodialysis treatments required for patients with acute renal failure, or number of units of blood administered to patients in need of transfusion. These findings suggest that the addition of pentoxifylline to artesunate therapy for severe malaria produces no evident clinical benefit.

Lack of effect of desipramine on the response to chloroquine of patients with chloroquine-resistant falciparum malaria.

The potential of desipramine to improve the efficacy of chloroquine against chloroquine-resistant Plasmodium falciparum in vivo in man was investigated. Fifty-three malaria patients were selected according to the criteria for the standard World Health Organization (WHO) in vivo test and were randomly divided in 2 groups. One group (n = 27) was given standard therapeutic doses of chloroquine (25 mg/kg body weight of base) and the other (n = 26) was given standard doses of chloroquine, as above, in combination with desipramine (1.3-2.8 mg/kg body weight) daily for 3 consecutive days. Standard WHO in vitro micro-tests were performed in parallel with the tests in vivo to provide chloroquine sensitivity patterns of the P. falciparum parasites. The results in vitro from both groups did not differ with regard to chloroquine sensitivity and the means of the pre-treatment parasite densities were similar. There was no apparent difference in parasite clearance in vivo between the 2 groups. This study provided no evidence for enhanced chloroquine efficacy in vivo through the use of desipramine in doses corresponding to the usual therapeutic range.

Susceptibility of Plasmodium falciparum in vitro to chloroquine, mefloquine, quinine and sulfadoxine/pyrimethamine in Somalia: relationships between the responses to the different drugs.

The susceptibilities of Plasmodium falciparum to chloroquine, mefloquine, quinine and sulfadoxine/pyrimethamine were investigated in Mogadishu in 1988, after chloroquine resistance had spread widely in Somalia. Possible correlations of the responses to these drugs were also investigated. Standard World Health Organization in vitro micro-tests were performed. Of 76 isolates tested for chloroquine susceptibility, 58 (76%) were resistant to the drug with mean EC50 and EC99 values of 1.06 x 10(-6) mol/litre and 10.44 x 10(-6) mol/litre of blood, respectively, indicating a high prevalence and degree of resistance. In contrast, all isolates tested were inhibited by mefloquine 3.2 x 10(-6) mol/litre of blood, quinine at 2.56 x (-6) mol/litre of blood-medium mixture, and sulfadoxine/pyrimethamine at 6.0/0.075 x 10(-6) mol/litre of blood-medium mixture, indicating full sensitivity to these 3 drugs. However, a significant positive correlation was found between responses to quinine and chloroquine and between those to quinine and mefloquine; the responses to chloroquine and mefloquine were not correlated. The results may suggest that sites responsible for the correlation are shared between quinine and chloroquine but not essentially between chloroquine and mefloquine. Thus the evolution of chloroquine resistance together with increased use of quinine treatment of P. falciparum may increase the risks of development of quinine resistance, whereas the development of mefloquine resistance would not be triggered by chloroquine resistance.

Susceptibility of Plasmodium falciparum to chloroquine and mefloquine in Somalia.

A field study was conducted in the Malable area, in Somalia, to assess the susceptibility of Plasmodium falciparum to chloroquine and mefloquine. The in vivo response of P. falciparum to standard therapeutic regimen of chloroquine was studied in 16 children (1-12 years) using the standard WHO in vivo field test. All subjects were parasite free by day 3 and no recrudescence occurred during a 7-d follow-up. In the 24 h micro in vitro tests for chloroquine, 29 of 39 tests performed were successful. Of the 29 isolates, 3 showed distinct resistance and 2 were borderline. The drug concentration yielding 99% (EC99) inhibition was 1.64 x 10(-6) M, indicating low grade resistance. For mefloquine, 11 of 20 tests gave interpretable results and were sensitive, although with some heterogeneity. The EC50 and EC99 of 0.24 x 10(-6) M and 1.31 x 10(-6) M respectively indicate sensitivity.

Plasmodium falciparum: susceptibility in vitro and in vivo to chloroquine and sulfadoxine-pyrimethamine in Ghanaian schoolchildren.

In Ghana, resistance of Plasmodium falciparum to chloroquine was observed for the first time in 1986. By the end of 1991 it had reached a high frequency and a substantial degree. A combined study in vivo and in vitro of the response of P. falciparum to chloroquine and sulfadoxine/pyrimethamine was carried out in Madina, Accra, in the coastal area of Ghana, late in 1991. 96 valid tests in vivo were performed with children and adolescents. There were 52 successful tests in vitro with chloroquine, and 52 with sulfadoxine/pyrimethamine. 45% of the chloroquine tests in vivo and 37% of the sulfadoxine/pyrimethamine tests in vivo indicated RII/RIII resistance. Results in vivo and in vitro were significantly correlated. The presence of RIII responses, 9% with chloroquine and 14% with sulfadoxine/pyrimethamine, indicates a need for third-line antimalarial drugs, the unregulated use of which may entail the risk of early and rapid occurrence of multi-resistance.

Chloroquine resistance of Plasmodium falciparum: a biological advantage?

The response in vitro of Plasmodium falciparum to chloroquine, mefloquine and quinine was studied in a hyperendemic peri-urban area of Accra, Ghana, during the fourth quarter of 1991, yielding a total of 159 valid tests. Schizont maturation in drug-free controls and effective chloroquine concentrations were strongly correlated. This was not seen with mefloquine or quinine. Higher mean parasitaemia in untreated oligo-symptomatic carriers of overtly chloroquine-resistant P. falciparum than in carriers of more sensitive parasites was another indication of higher viability and biological advantage of chloroquine-resistant P. falciparum that may conceivably have clinical implications.

An epidemic of Plasmodium falciparum malaria in Balcad, Somalia, and its causation.

The causative factors of an epidemic of falciparum malaria were investigated in Balcad, Somalia, a town with previously low malaria transmission, where malaria incidence rose more than twenty-fold between 1986 and 1988. The emergence of chloroquine resistance, accelerated by high drug pressure, low herd immunity and favourable meteorological conditions were identified as major causes of the epidemic. Chloroquine resistance of grades RII and RIII was first observed in Balcad in 1987 and rapidly increased to 72% of the Plasmodium falciparum infections in 1988. In the absence of alternative treatment, resistance resulted in the accumulation of a massive infective reservoir and therefore increased malaria transmission, associated with intensive clinical symptomatology. The advent of chloroquine resistance was less violent in the area of Malable, where malaria is stable and communal immunity higher than in Balcad.

The changing pattern of Plasmodium falciparum susceptibility to chloroquine but not to mefloquine in a mesoendemic area of Somalia.

The response of Plasmodium falciparum to chloroquine and mefloquine was investigated in a mesoendemic area of Somalia from 1986 to 1989. Serial in vivo field tests for chloroquine sensitivity were performed and the sensitivity in vitro to chloroquine and mefloquine was evaluated using the standard WHO in vitro microtest. Chloroquine treatment in vivo (25 mg base/kg body weight) resulted in parasite clearance in all patients within 7 d (S/RI) in 1986, while 17%, 19% and 30% RII/RIII responses were found in 1987, 1988 and 1989 respectively. There was consistent increase of parasite clearance time of the S/RI cases in all years. The sensitivity study in vitro in 1986 showed a low degree of chloroquine resistance in 3 of 29 isolates tested and a mean 50% effective dose (EC50) and EC99 of 0.34 x 10(-6) M and 1.99 x 10(-6) M, respectively. In contrast, in 1989, 12 of the 19 isolates tested were resistant to chloroquine. The mean EC50 and EC99 values had increased to 0.78 x 10(-6) M and 7.50 x 10(-6) M respectively. The data in vivo and in vitro indicate a rapid increase of chloroquine resistance both in frequency and degree. All isolates tested in 1986 and 1989 were fully inhibited by mefloquine at 3.2 x 10(-6) M, suggesting full sensitivity. Thus, increased resistance of P. falciparum to chloroquine did not significantly influence the sensitivity pattern of mefloquine.

High level of resistance of Plasmodium falciparum to sulfadoxine-pyrimethamine in children in Tanzania.

In many areas of tropical Africa affected by chloroquine-resistant Plasmodium falciparum, a combination of sulfadoxine and pyrimethamine (S-P) is used for alternative medication, especially in young children. In Magoda village in Muheza District, north-eastern Tanzania, 38 children 1-10 years of age were enrolled in a therapeutic study of S-P in July 1994. All had monoinfections of P. falciparum and an asexual parasite count of 1000-80,000/microL of blood. S-P was given as a single dose corresponding to 0.8-1.4 mg pyrimethamine/kg body weight. Of the 38 children followed up to day 7, 10 showed an S/RI response, 26 an RII response, and 2 an RIII response. Older children had lower pre-treatment parasitaemia and a better therapeutic response than younger children. Among the various contributory factors responsible for the poor therapeutic result, drug pressure from a prophylactic intervention with weekly dapsone-pyrimethamine between May 1993 and May 1994 seems to have been the most important.

In vitro assessment of sensitivity of Plasmodium falciparum to chloroquine and mefloquine in Ghana.

An in vitro study of sensitivity of Plasmodium falciparum to chloroquine and mefloquine in Ghana is described. Results of 60 short-term cultures from 36 patients are evaluated. No sign of chloroquine resistance was found as all microtests showed complete inhibition of maturation at a level of 0.8 X 10(-6) M. For mefloquine schizont maturation was seen at higher levels of the drug. However, the estimated EC99, with 2.2830 X 10(-6) M is probably within the range of sensitivity.

Differences in drug response of Plasmodium falciparum within an area of the Amazon region.

Clinical characteristics of patients with falciparum malaria, as well as sensitivity of Plasmodium falciparum to chloroquine and mefloquine, were investigated in 2 distinct strata within the same geographical area of the Amazon Basin. One stratum was the population living along the road, the other that living along the river, both near Rio Branco, capital of Acre State, Brazil. The clinical features did not differ between the 2 strata. Full in vitro sensitivity of P. falciparum to mefloquine was observed in both areas. However, significant differences in chloroquine sensitivity were observed between the 2 strata. EC50 values for chloroquine were 0.8484 mumol/litre for parasite isolates from the road stratum (E) and 0.4638 mumol/litre for parasite isolates from the river stratum (R). EC90 values were 2.8095 and 1.2549 mumol/litre in strata 'E' and 'R', respectively. Continuous drug pressure over years in area 'E' and relatively low drug pressure in area 'R' were presumably responsible for these differences.

Reassessment of the resistance of Plasmodium falciparum to chloroquine in Gabon: implications for the validity of tests in vitro vs. in vivo.

Increasing resistance of Plasmodium falciparum to antimalarial drugs presents a major risk factor for people living in endemic areas of tropical Africa. In Lambaréné, Gabon, regular surveillance of chloroquine sensitivity of P. falciparum in vitro has been carried out since 1992 using the WHO standard microtest. Results indicated that from 1994 onwards chloroquine resistance in vitro decreased significantly and that by 2000, about 70% of parasite isolates seemed to be sensitive to chloroquine in vitro. In 2001, we conducted a clinical study to reassess the efficacy of chloroquine in vivo for the treatment of uncomplicated P. falciparum malaria. Twenty-six patients aged 4-15 years were included in this study. Most unexpectedly, the study demonstrated high-grade resistance to chloroquine in vivo (failure rate on day 28 of 100%). As a consequence, tests of parasite susceptibility to chloroquine in vitro were repeated using the same protocol except for the replacement of previously used commercially available predosed WHO culture plates by independently dosed plates. All tested P. falciparum isolates were highly resistant to chloroquine, correlating well with our clinical findings. We concluded that high level resistance of P. falciparum to chloroquine persists in the study area. Neglect or absence of quality controls of essential test material can lead to invalid study results and wrong conclusions and should always be suspected in the case of major fluctuations in the sensitivity patterns of an antimalarial drug in vitro. In addition, our results highlight the supreme value of tests in vivo in providing reliable estimates of the efficacy of an antimalarial in a specific area.