These 3 authors contributed equally to this study.
Pyronaridine and artesunate have been shown to be effective in falciparum malaria treatment. However, pyronaridine is rarely used in Hainan Island clinically, and artesunate is not widely used as a therapeutic agent. Instead, conventional antimalarial drugs, chloroquine and piperaquine, are used, explaining the emergence of chloroquine-resistant
In recent years, although the morbidity and mortality of malaria in china were significantly decreased [
Dihydroartemisinin is a derivative of artemisinin with a strong therapeutic effect against falciparum malaria. Dihydroartemisinin/piperaquine phosphate (DUO-COTECXIN) is a new antimalarial drug developed and produced in China, in accordance with the guidelines of WHO. Pyronaridine and artesunate have been shown to be effective in falciparum malaria treatment. However, pyronaridine is rarely used in Hainan Island clinically, and artesunate is not widely used as a therapeutic agent. Instead, conventional antimalarial drugs such as chloroquine and piperaquine were used, explaining the emergence of chloroquine-resistant
The drugs, patients, and methods in this study were reviewed and approved by the Ethics Committee of the Center for Disease Control in Hainan Province, China.
Hainan malaria endemic areas have been divided into 3 layers; higher, middle, and lower by corresponding to their incidence. In this present study, 2 counties were selected from high endemic areas and 2 middle areas, respectively. Four cities and counties (Dongfang City, Sanya City, Baisha Country, and Lingshui County) with a high incidence of falciparum malaria in Hainan Island were selected by stratified randomization (
Patients aged between 4 and 70 years were selected ensuring that the microscopic density of asexual parasites was 1,000-8,000 parasites per μl of blood. The patients were required to have no history of antimalarial drug usage 15 days prior to the onset, and to be negative for urinary 4-aminoquinoline. The body temperature of these patients was ≥37.5˚C or manifesting fever within 24 hr. Patients with severe heart, kidney, liver, and hematopoietic diseases or severe malnutrition, or patients who had taken antimalarial drugs 15 days prior to the onset, pregnant and lactating women, those with a history of allergy for DUO-COTECXIN, and critical cases, were excluded from this survey. All the patients voluntarily participated in this survey and provided signed informed consent.
The in vivo sensitivity of
The in vitro test was performed using a modification of the 96-well microplate technique recommended by WHO [
Patients involved in the in vivo test were hospitalized for observation and discharged after asexual parasite clearance, body temperature recovery, and symptom relief, were followed up to 28 day. The resistance rate, inhibitory dose 50 (IC50) and the mean concentration for complete inhibition (CIMC) of schizont formation were measured in vitro, to observe the treatment effect.
The time of parasite clearance was defined as the time until first of 3 consecutive counts of negative parasites after administration. The mean parasite clearance was calculated using the arithmetic average method. The axillary temperature was measured every 4 hr before and after drug administration. Fever clearance was defined as the time for the body temperature to return to normal over 2 consecutive times. The mean fever clearance time was calculated using the arithmetic average. A cure was indicated if asexual parasite clearance was observed in patients’ blood test 7 days after drug administration without relapse after 28 days. The cure rate was expressed as a percentage. Parasite recrudescence (expressed as a percentage) was monitored on days 7, 14, 21, and 28 after the drug administration. The adverse reaction rate was expressed as a percentage. IC50, the dose required to inhibit half of the tested subjects, was calculated by ED50 regression. CIMC, the concentration required to inhibit all the tested subjects, was calculated using the arithmetic average method. Drug resistance rate was expressed as a percentage.
Evaluation of the in vivo test was based on the WHO evaluation criteria of susceptibility testing for
A successful in vitro test shows more than 10% rate of schizont (with more than 3 nuclei) development among the parasites in the control well of the test plates (provided by National Institute of Parasitic Diseases, Chinese Center for Diseases Control and Prevention). If the parasites developed to schizonts in the wells containing 0.000008 μmol chloroquine, 0.000032 μmol piperaquine, 0.000008 μmol pyronaridine, and 0.000004 μmol artesunate, respectively.
The statistical analysis was performed using software Excel and SPSS13.0. The rank sum test was conducted for data analysis and the chi-square test was used to compare the data.
A total of 58 patients were admitted to this test, and 46 patients were finally enrolled, among which 34 were from Dongfang City (Jiangbian Township), 6 were from Sanya City (Tianya and Fenghuang Township), 4 were from Baisha Country (Qifang and Xishui Township), and 2 were from Lingshui Country (Zuguan Township). The eligible patients included 37 males and 9 females aged from 5 to 67 years, with an average age of 27.8±15.7 years. Fourteen patients were ≤15 years and 32 were ≥16 years. Before administration, patients’ body temperature ranged from 35-40˚C, with an average of 38.5±1.0˚C. The parasite density was 1,000-149,280 parasites per μl blood, and the mean density was 16,663±24,134 parasites per μl blood.
Among 6 patients from Sanya City, 1 was a case of
Among 46 patients, the mean fever clearance time was 22.5±10.6 hr and the mean parasite clearance time was 27.3±12.2 hr. The mean fever clearance time for males was 23.6±11.2 hr and 21.2±7.8 hr for females. The difference of mean parasite clearance time between men and women was insignificant, 27.6±12.6 hr and 27.8±14.9 hr, respectively. Based on the rank sum test, the mean fever clearance time (
The high fever group was defined as the body temperature was>39˚C before administration, the lower group was defined as the body temperature was ≤39˚C but above normal temperature. The mean fever clearance time for the high fever group was 28.8±14.8 hr, which was 21.9±8.4 hr for the low fever group. The mean parasite clearance times for the 2 groups were similar. The difference between the mean fever clearance time and the mean parasite clearance time at different body temperatures before drug administration was statistically not significant, by the rank sum test. The results are shown in
In terms of the parasite density, the mean fever clearance time for the group with a parasites density of ≤12,000 parasites per μl blood was 23.2±9.3 hr, which was 18.7±5.2 hr for the group with a density of 12,000 parasites per μl blood. The mean parasite clearance times in the 2 groups were similar, 26.5±14.8 hr and 26.8±6.4 hr, respectively. The difference between the mean fever clearance time (
One day before drug administration, 1 patient had symptoms of headache, dizziness, nausea, and vomiting, which were not significantly worsened after the drug administration. These may not be related to the drug. However, 2 patients manifested the above symptoms, which may be related to the drug and disappeared after the parasite clearance and the temperature returned to normal.
Total 32 patients were observed in this test. The resistance rate of
The antimalarial effect of the 4 drugs increased with the concentration. The relationship between the drug concentration and number of inhibited cases was similar to a S-curve (
Statistical differences in CIMC were observed with these 4 antimalarial drugs (
Dihydroartemisinin has a curative effect for falciparum malaria better than other antimalarial drugs. The inadequacy of the course is longer, and has recrudescence [
In 1974, resistance of
In the present study, artesunate showed the lowest effective concentration and the lowest concentration of complete inhibition while chloroquine had the highest. The relationship of antimalarial effect between the 4 drugs varied as follows: artesunate>pyronaridine>piperaquine>chloroquine. Artemisinin-based drugs showed a strong dose-dependent lethal effect against
According to a previous study, antimalarial drugs had mild adverse reactions, with the adverse reaction rates of chloroquine 27% and of piperaquine 17% [
This study received a financial support from the Hainan Provincial Scientific Research grant, China (grant nos. 813251 and 310174). We would like to thank the staff from Dongfang CDC, Sanya CDC, Baisha CDC, and Lingshui CDC, China.
The authors declare that they have no competing interests. There is also no conflict of interest with the drug (DUOCOTECXIN) producer.
Map of China and Hainan Island and survey sites.
Antimalarial drugs: dose-response curve.
Comparative drug efficacy by age
Index | 16 | < 16 | Statistics | |
---|---|---|---|---|
Mean fever clearance time (hr) | 25.4 ± 11.0 | 17.7 ± 6.8 | Z = -1.920 | 0.06 |
Mean parasite clearance time (hr) | 27.6 ± 11.9 | 27.6 ± 13.9 | Z = -0.018 | 0.98 |
Comparative drug efficacy by body temperature
Index | > 39˚C | ≤ 39˚C | Statistics | |
---|---|---|---|---|
Mean fever clearance time (hr) | 28.8 ± 14.8 | 21.9 ± 8.4 | Z = -0.413 | 0.68 |
Mean parasite clearance time (hr) | 25.5 ± 6.9 | 27.4 ± 16.6 | Z = -0.940 | 0.35 |
Comparative drug efficacy by different parasitemia
Index | ≤ 1.2 × 104 p/μl | >1.2 × 104 p/μl | Statistics | |
---|---|---|---|---|
Mean fever clearance time | 23.2 ± 9.3 | 18.7 ± 5.2 | Z = -1.820 | 0.069 |
Mean parasite clearance time | 26.5 ± 14.8 | 26.8 ± 6.4 | Z = -1.210 | 0.23 |
Resistance rate in vitro
Drugs | Cases tested | Cases with resistance |
Resistance rate (%) |
---|---|---|---|
Chloroquine | 32 | 23 | 71.9 |
Piperaquine | 32 | 13 | 40.6 |
Pyronaridine | 32 | 4 | 12.5 |
Artesunate | 32 | 0 | 0 |
The number was counted when the case did not belong to sensitivity level after administration.
Results of IC 50 in vitro
Drugs | Cases tested | Cases with resistance | IC 50/(× 10-6 mol/L) |
---|---|---|---|
Chloroquine | 32 | 23 | 3.77 ± 1.40 |
Piperaquine | 32 | 13 | 2.09 ± 3.30 |
Pyronaridine | 32 | 4 | 0.09 ± 0.05 |
Artesunate | 32 | 0 | 0.05 ± 0.01 |
In vitro results of CIMC
Drugs | Cases tested | Cases with resistance | CCIMC/(× 10-6 mol/L) |
---|---|---|---|
Chloroquine | 32 | 23 | 5.60 ± 5.64 |
Piperaquine | 32 | 13 | 9.26 ± 13.67 |
Pyronaridine | 32 | 4 | 0.55 ± 1.02 |
Artesunate | 32 | 0 | 0.07 ± 0.04 |