新型荧光光源吖啶橙梯度染色法检测伯氏疟原虫的研究

探讨新型荧光光源吖啶橙(AO)梯度染色法检测伯氏疟原虫的效率和实用性.对经血液腹腔接种传代感染5d的伯氏疟昆明小鼠(60只)以红细胞的疟原虫寄生率计算法计算原虫密度,并用正常小鼠新鲜血10倍倍比稀释其尾血或眼球血,同时推制薄血片.用新型荧光光源AO法和瑞氏法染色后高倍镜检,记录每片发现三次原虫的平均时间,5min观察300个视野未发现疟原虫者为阴性.本AO染色法、瑞氏染色法能检出的伯氏疟原虫的最低原虫密度分别为11虫/μL、19虫/μL.当原虫密度在一定范围内时,两法检测发现疟原虫的时间有差别,前者比后者快.原虫密度适中的稀释倍数组原虫密度与各法检出的时间为负相关.不同稀释倍数组两法检出时间均为正相关.新型荧光光源AO梯度染色法是检测人体疟原虫的一种简便、快速、敏感、实用的方法,可推广作为疟疾流行病学调查及后期监测的手段.     

Cytoadherence of knobless Plasmodium falciparum-infected erythrocytes and its inhibition by a human monoclonal antibody

Red blood cells infected with mature stages of the malaria parasite Plasmodium falciparum bind to the endothelial lining of capillaries and venules. This sequestration is important for the survival of the parasite but may have severe consequences for the host. For example, it is involved in the causation of cerebral malaria which carries 25% mortality. Knob-like protrusions present on the surface of infected erythrocytes have been considered necessary but not sufficient for this cytoadherence. Here we describe the adhesion to endothelial cells of infected erythrocytes which do not have knobs. A human monoclonal antibody (33G2) which was specific for an epitope containing regularly spaced dimers of glutamic acid present in the repeated amino-acid sequences of some defined P. falciparum antigens was found to inhibit cyto-adherence and may therefore be an important reagent for elucidating the molecular basis of parasite sequestration.     

Several alleles of the multidrug-resistance gene are closely linked to chloroquine resistance in Plasmodium falciparum

The lethal form of human malaria caused by Plasmodium falciparum is virtually uncontrollable in many areas because of the development of drug resistance, in particular chloroquine resistance (CQR). CQR is biologically similar to the multiple drug resistance phenotype (MDR) of mammalian tumour cells, as both involve expulsion of drug from the cell and both can be reversed by calcium channel antagonists. A homologue (pfmdr1) of the mammalian multidrug resistance gene has been implicated in CQR because it is amplified in some CQR isolates of P. falciparum as is an mdr gene in MDR tumour cells. We show here that the complete sequences of pfmdr1 genes from 2 CQ sensitive (CQS) P. falciparum isolates are identical. In 5 CQR isolates, 1-4 key nucleotide differences resulted in amino acid substitutions. On the basis of these substitutions, we have correctly predicted the CQS/CQR status of a further 34 out of 36 isolates. This is a paradox as CQR arises much less frequently than would be predicted if single point mutations were sufficient. We conclude that a mutated pfmdr1 gene is one of at least two mutated genes required for CQR.     

Chloroquine resistance not linked to mdr-like genes in a Plasmodium falciparum cross

Chloroquine is thought to act against falciparum malaria by accumulating in the acid vesicles of the parasite and interfering with their function. Parasites resistant to chloroquine expel the drug rapidly in an unaltered form, thereby reducing levels of accumulation in the vesicles. The discovery that verapamil partially reverses chloroquine resistance in vitro led to the proposal that efflux may involve an ATP-driven P-glycoprotein pump similar to that in mammalian multidrug-resistant (mdr) tumor cell lines. Indeed, Plasmodium falciparum contains at least two mdr-like genes, one of which has been suggested to confer the chloroquine resistant (CQR) phenotype. To determine if either of these genes is linked to chloroquine resistance, we performed a genetic cross between CQR and chloroquine-susceptible (CQS) clones of P. falciparum. Examination of 16 independent recombinant progeny indicated that the rapid efflux phenotype is controlled by a single gene or a closely linked group of genes. But, there was no linkage between the rapid efflux, CQR phenotype and either of the mdr-like P. falciparum genes or amplification of those genes. These data indicate that the genetic locus governing chloroquine efflux and resistance is independent of the known mdr-like genes.