Plasmodium vivax: Isotopic, PicoGreen, and microscopic assays for measuring chloroquine sensitivity in fresh and cryopreserved isolates

In vitro susceptibility tests provide information on the intrinsic response of Plasmodium vivax to antimalarials, free from confounding factors such as host immunity or relapse. This study examined the utility of radioisotope and PicoGreen assays as alternatives to the traditional microscopic examination for assessing response of P. vivax to antimalarial drugs. There was no significant difference in the mean chloroquine IC50 of P. vivax (n = 40) as determined by the microscopic (33.4 ng/ml), isotopic (33.6 ng/ml), and PicoGreen (39.1 ng/ml) assays, respectively (F = 0.239, df = 2, 51, and p = 0.788). However measurement of IC50s by the microscopic method was slightly more successful in producing valid assays (57%), compared to the isotopic (32.5%) and PicoGreen (45.5%) methods. In a paired comparison of 20 fresh and cryopreserved isolates as examined by the microscopic method, there were no significant differences between the mean IC50 responses (T = 1.58, df = 15, and p = 0.34). Detailed methodologies for the short time culture of field and cryopreserved P. vivax are described. Although the microscopic in vitro assay provides a useful method for characterizing the drug susceptibility phenotype of P. vivax isolates, its utility is limited by a laborious methodology and need for highly skilled microscopists. Future efforts should focus on further development of high throughput assays such as the PicoGreen assay as described in this study

Plasmodium vivax: isotopic, PicoGreen, and microscopic assays for measuring chloroquine sensitivity in fresh and cryopreserved isolates.

In vitro susceptibility tests provide information on the intrinsic response of Plasmodium vivax to antimalarials, free from confounding factors such as host immunity or relapse. This study examined the utility of radioisotope and PicoGreen assays as alternatives to the traditional microscopic examination for assessing response of P. vivax to antimalarial drugs. There was no significant difference in the mean chloroquine IC(50) of P. vivax (n=40) as determined by the microscopic (33.4 ng/ml), isotopic (33.6 ng/ml), and PicoGreen (39.1 ng/ml) assays, respectively (F=0.239, df=2, 51, and p=0.788). However measurement of IC(50)s by the microscopic method was slightly more successful in producing valid assays (57%), compared to the isotopic (32.5%) and PicoGreen (45.5%) methods. In a paired comparison of 20 fresh and cryopreserved isolates as examined by the microscopic method, there were no significant differences between the mean IC(50) responses (T=1.58, df=15, and p=0.34). Detailed methodologies for the short time culture of field and cryopreserved P. vivax are described. Although the microscopic in vitro assay provides a useful method for characterizing the drug susceptibility phenotype of P. vivax isolates, its utility is limited by a laborious methodology and need for highly skilled microscopists. Future efforts should focus on further development of high throughput assays such as the PicoGreen assay as described in this study

Physiological properties of human diaphragm muscle fibres and the effect of chronic obstructive pulmonary disease

The contractile and actomyosin ATPase properties of single fibres were examined in human diaphragm muscle obtained from patients with and without chronic obstructive pulmonary disease (COPD). Costal diaphragm biopsies were taken from five patients without evidence of COPD and from 11 age-matched individuals with varying degrees of the disease. Our aim was to establish whether changes in contractile properties of COPD diaphragm could be fully explained by the previously documented shift towards a greater proportion of type I myosin heavy chain isoform in COPD. The relative proportion of type I diaphragm fibres from non-COPD and COPD patients was measured by gel electrophoresis, and was negatively correlated with FEV1 over the full range of values investigated. There was also significant atrophy of the type I fibre population in COPD diaphragms. Isometric tension was similar among the fibre types and between the COPD and non-COPD patients. The intrinsic energetic properties of diaphragm fibres were examined by monitoring the time-resolved actomyosin ATPase activity in COPD and non-COPD fibres that produced similar isometric forces. The isometric ATPase rate in COPD fibres was reduced to 50% of the rate in non-COPD fibres; hence, the cost of isometric contraction in type I and type IIA COPD fibres was reduced to between one-third and one-half of the tension cost calculated for non-COPD fibres. The rate of force development in type I COPD fibres was reduced to 50% of the rate seen in non-COPD type-I fibres. No difference in the rate of ATP consumption between COPD and non-COPD fibres was evident during isovelocity shortening. These data extend previous findings showing that aspects of breathing mechanics during progressive COPD are associated with remodelling of the diaphragm fibre-type distribution; on top of the increase in type I fibres there are fibre-specific reductions in force development rate (type I fibres) and ATPase rate that are consistent with the impairment of cross-bridge cycling kinetics