| Objectives:1. To determine the in vitro ICso values of Plasmodium falciparum collected in2007-2011from Wa State and in2011from Kachin State, Myanmar, to five antimalarial drugs pyronaridine, naphthoquine phosphate, mefloquine, lumefantrine and pyrimethamine and to predict the trend of drug susceptibility in this area.2. To determine the sequence polymorphism of pfindrl by nested PCR and sequencing and compare the codon mutations between the parasite populations collected from Wa and Kachin states.3. To further refine conditions for the4℃cold storage synchronization method.Methods:1. By genotyping two polymorphic genes mspl and msp2,23P. falciparum isolates with multiclonal infections from Wa State and3isolates with monoclonal infection from Kachin State were selected and cultured. Based on competition of parasite strains during in vitro culture, the time for the mixed infections to become monocloncal infection was determined by genotyping mspl and msp2. Drug sensitivity to five antimalarial drugs was determined for monoclonal infections.2. Selected monoclonal isolates were cultured to>70%ring stage and parasitemia of>1%. For drug assay, the microculture plates were predosed with serially diluted antimalarial drugs. The culture was adjusted to a parasitemia of0.5%and hematocrit to2%, added to culture plates, incubated at37℃in a5%CO2incubator for72h, and frozen at-20℃. Within one week, SYBR Green I assay was performed to determine the IC50values. Sensitivities of23Wa isolates and3Kachin isolates to the5afore-mentioned antimalarials were determined. For accuracy of the assay, an international reference strain (3D7) was included and each sample was assayed in3biological replicates. Growth inhibition rates of the drugs were calculated; the IC50value, its95%confidence interval, and drug-response curve were determined using Prism5.0. Drug sensitivity was defined as "sensitive" if the IC50value is less than or equal to that of3D7, and "resistant" if the IC50value is higher that of3D7. The drug sensitivity data in each year was used to estimate the trend of drug sensitivity for the Wa state.3. Pfmdrl of these parasite isolates were sequenced to identify mutations and to compare the parasite populations from Wa and Kachin states.4. Schizonts, obtained using the Percoll-Sorbitol gradient method, were tested for their tolerance of4℃treatment. The4℃storage synchronization method was compared using4laboratory strains and4clinical isolates. The percentage of ring stage before and after4℃storage was recorded and compared. The3D7cultures at different ratios of ring-stage parasites (30-50%,50-70%,>70%) were treated with the4℃refrigeration method, and the ratios of ring-stage parasites after the treatments were compared.Results:1. Of the23Wa P. falciparum isolates with multiclonal infections,5,8,8,1, and1isolate became monoclonal after continuous culture20,30,40,50, and90days, respectively.2. The in vitro IC50values of the23Wa isolates and3Kachin isolates of P. falciparum to the five antimalarial drugs were3.2±1.8nM for pyronaridine,5.9±4.0nM for naphthoquine phosphate,51.6±33.7for mefloquine,2.4±1.5for lumefantrine, and2907.1±2109.3nM for pyrimethamine. The sensitivity rates for the five drugs were different (P<0.005). Parasites were highly resistant to pyrimethamine, whereas they remained sensitive to the other four drugs. Particularly in2010, the resistance rate rose to100%. The3Kachin isolates collected in2011were still sensitive to the five antimalarials and only a few showed slightly decreased sensitivity. Subsequently we made multiple comparisons of the sensitivity rate among the Wa isolates between the years, and found no significant differences between the years for pyronaridine, mefloquine and pyrimethamine, whereas there were significant differences between the years for naphthoquine phosphate and lumefantrine. However, significant difference was only limited to one year, and sensitivity rates of the other three years remained100%.3. Of the38isolates sequenced for pfmdrl (a960bp fragment from two isolates were not successfully sequenced),21and17were from Wa and Kachin states. Among them,12Wa and8Kachin isolates had mutations. The86Y was found in8isolates (21.1%), of which7from Wa and1from Kachin. The130K mutation was identified in3Wa isolates (7.9%). The184F mutation was found in8isolates (21.1%), of which6were from Kachin and2from Wa. The1042D mutation were present in1isolate (2.8%) from Wa State. A new mutation N173K was found in an isolate (F08B35) from Kachin. One sample (WBLH5) contained mutations at two positions Y184F/N1042D. Of the sequenced samples,19isolates contained a single mutation, whereas18isolates were of wild type. In terms of the mutation frequencies, there was no significant difference between Wa and Kachin parasite populations (x2=0.38,0.5<P<0.75).4. The4℃refrigeration experiments showed that schizonts could not tolerate the cold treatment. For cultures with an initial ring-stage ratio of37.5-44.1%, storage at4℃for9-24h resulted in more synchronous cultures with ring-stage ratios of77.0-85.6%. In addition, this synchronization method is highly repeatable. Statistical analysis showed that ring-stage ratios of both laboratory clones and clinical isolates after4℃synchronization were not significantly different (P=0.194). For3D7cultures with an initial ring-stage ratio of30-50%,50-70%, and>70%, the resulting ring-stage ratios after4℃synchronization were not significantly different (F=1.990, P=0.217, ANOVA).Conclusions:1. Multiclonal infections gradually changed into monoclonal after continuous in vitro culture, and mostly occurred within40days.2. Wa isolates of P. falciparum (2017-2011) were sensitive to pyronaridine, naphthoquine phosphate and mefloquine, but the sensitivity decreased over the years. Except one isolate in2010, parasites were sensitive to lumefantrine. Parasites were highly resistant to pyrimethamine and the resistance rate in2010reached100%. P. falciparum from Kachin State (2011) were sensitive to five antimalarials, and a few isolates showed slightly decreased sensitivity to some antimalarials.3. The prevalence of mutations in pfmdrl of isolates was not significantly different between Wa and Kachin parasite populations. The order of the frequencies of mutations is N86Y=Y184F>G130K>N1042D>N173K.4. Schizonts were killed by4℃refrigeration. The4℃refrigeration method is simple to perform, and reduces the possibility of contamination during the experiment process. P. falciparum treated by this method can meet the specified conditions set for the drug sensitivity assay. When compared with the5%sorbitol synchronization method, the results were similar. This method was equally effective for synchronizing laboratory strains and field isolates. Parasites with ring-stage ratios of>30%could also achieve similar levels of synchrony after4℃refrigeration. |
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