| 1. Objective1.1 Observation of the experimental group (Naphthoquine combination with Azithromycin) and the control group (control group:single Naphthoquine use) of Plasmodium falciparum in vitro drug stimulation (pressure), in order to sensitivity and its changes in speed.1.2 With the WHO recommended Rieckmann in vitro determination of the sensitivity of the different stages Plasmodium falciparum to Naphthoquine and Azithromycin, Naphthoquine/Azithromycin in control group and experimental group to calculate the amount of half inhibition (IC50).1.3 Described the characteristics of the action for the two drugs combination in vitro and evaluated the two drugs combination whether or not delay resistance or potential. Combination therapy may provide a scientific basis for the clinical treatment malaria programs.2. Methods2.1 Strain and culture:Culture the Plasmodium falciparum strain selected from samples by Trager method in vitro2.2 Observation the effect of drug:The resistant Plasmodium falciparum strains were stimulated by corresponding drugs with increasing concentrations disconnectedly.2.3 In vitro assay:Rieckmann's in vitro micro-technique was used for assay. The concentration of in vitro cultured Plasmodium falciparum strain were diluted to 20,000-60,000/μ1 with culture medium, which was added to pre-prepared drug plates at 50μ/well after synchronization. Selected Plasmodium falciparum cultured in one of control wells after 20-24hours'culture in 37℃, the harvested time was determined by the growth of Plasmodium falciparum. Based on the number of schizonts in control well, counted the inhibitive ratio of schizonts in each well, calculated half inhibitive dose (IC50) of every drug to parasite by ICEstimator software.2.4 Estimated standard:When IC50 of Plasmodium falciparmst to artesunat was 5 time than that of parent, then we consider corresponding P. falciparum as artesunate-resistant strain. During the same period of resistance culturing in vitro, if IC50 value of P. falciparumst to artesunat combined with clindamycin was 5 times than that of parent, which could be deemed that the P. falciparumst had resistance to the tow drugs; otherwise, the IC50 did not rise obviously or much more lower than that of parent, however, artesunate combined with clindamycin exhibit an apparent synergistic effect in vitro.1. Results3.1 Frequency, concentration and results of drug touching. The control group falciparum contact Naphthoquine 150 days, total contact drug 15 times, drug concentration tools 2.56 nmol/L five times,160nmol/L twice,5,10,20,40,80,320,625,1280nmol/L each time respectively. Contact drugs before, sensitive strains (parental department) of IC50 of 2.11 nmol/L, drug stimulates 15 times, after the IC50 to 65.47 nmol/L. Approximately dictated protozoon 31 times. Experimental falciparum contact Naphthoquine/Azithromycin 120 days, total contact drug nineteen times, drug concentration respectively for 0.31/0.256 nmol/L eight,2.49/2.135 nmol/L five times,0.625/0.53,4.98/4.2, 9.735/8.34,19.47/16.685,38.94/33.375,77.88/66.755nmol/L each time. Contact drugs before, sensitive strains (parental department) of IC50 for 0.18 nmol/L,0.15 nmol/L, drug stimulates nineteen times and its IC50 to 2.68 nmol/L,2.89nmol/L respectively, approximately dictated protozoon 16.1 times,17.9times.3.2 Sensitivity of P. falciparum before adding drugs. In P. falciparum not contact with drugs, before Naphthoquine determination board, Azithromycin determination board and Naphthoquine/Azithromycin composite plate determination plasmodium P. falciparum determination sensitivity, in a single Naphthoquine determination plate IC50 of 2. llnmol/L, In a single Azithromycin determination plate measured IC50 for 3.21nmol/L, In Naphthoquine/Azithromycin composite determination boards measured with quetiapine alpha-naphthol of Azithromycin IC50 were detected 0.15 nmol/L. A single measurementboards Naphthoquine IC50) is the determination of the composite board 11.7 times (2.11/0.18). The determination of Azithromycin IC50 is composite plate 21.4 times (3.21/0.15).3.3 Sensitivity of P. falciparum before and after adding Naphthoquine i.n contrast group The control group in a single contact Naphthoquine plasmodium falciparum IC50 for a single after contact with Naphthoquine before 31.0(65.47/2.11) times. The control group in a single contact Naphthoquine plasmodium falciparum to the sensitivity of the question of Azithromycin is single contact Naphthoquine 21.4 (65.58/3.21) times.3.4 Sensitivity of P. falciparum before and after adding Naphthoquine/ Azithromycin in test group Experimental Plasmodium falciparumin contact Naphthoquine/Azithromycin after contact with drugs for IC50 before 16.8(35.47/2.11) times. Experimental plasmodium falciparum in contact Naphthoquine/Azithromycin after contact with drugs for IC50 before 15.6 (50.04/3.21) times.3.5 Compare the effect of artesunate clindamycin combination and aretsunate single used to P. falciparum The control group in contact with drugs in a single Naphthoquine IC50 for the determination of board 65.47 nmol/L, in a single Azithromycin determination boards measured for IC50 68.58nmol/L, in Naphthoquine/Azithromycin compound respectively in the determination of board IC50 10.34nmol/L,8.46nmol/L. After the experimental drug in contact in a single Naphthoquine determination for the IC50 35.47nmol/L plate in a single/L, determination of Azithromycin plate measured for IC50 50.04nmol/L, in Naphthoquine/Azithromycin composite board of determination IC50 respectively 2.89 nmol/L,2.68nmol/L4. Conclusions4.1 Falciparum parasites in a single contact Naphthoquine after a period of time can produce resistance, and its IC50 far outclass dictated by more than 5 times, further confirmed that the artificial cultivation method can cultivate anti Naphthoquine resistant falciparum strains.4.2 Drug tolerability growth on experimental falciparum far slower than control group, so it hints Naphthoquine and Azithromycin two drugs coupled have obvious synergy and delay the resistance and deserves further study. 4.3 Group of drug falciparum tolerated growth far slower than the control group, the former in drug stimulates 120 acquired Naphthoquine, Azithromycin, Naphthoquine/Azithromycin IC50 were up by 16.8 times of 15.5 times, and 16.1/17.9 times, the latter stimulate 120 days Naphthoquine, Azithromycin, Naphthoquine/Azithromycin IC50 were up by the victims times,31.0 times,21.4times,57.4/56.4 times, suggesting Naphthoquine and Azithromycin two drugs have obvious delay the resistance role. |
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