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An Evaluation of the Criteria of Cure in Experimental Leishmaniasis of the Golden Hamster^1,2,

In the present study, several methods for assaying the chemotherapeutic activity of drugs in experimental leishmaniasis have been investigated. This results indicate that these methods fall in the following order of decreasing sensitivity: culture of a) material obtained by splenic puncture, b) macerated spleen, c) macerated liver, d) intact spleen, the microscopic examination of e) splenic smears, and f) the culture of intact liver.

The fact that splenic cultures were regularly more sensitive than similar cultures of liver in detecting treatment failures may be attributed to the greater number of organisms in the spleen as well as their microscopically more uniform distribution. Splenic tissue in excess was found to inhibit the growth of the organisms; and in consequence, cultures of macerated spleen in which the final concentration of splenic tissue was 1:5,000 proved significantly more sensitive than similar cultures of macerated spleen containing 1 per cent splenic tissue. The small amount of spleen obtained by puncture and aspiration through a 20-gauge needle proved to be in the optimal range, and constituted a simple and reliable routine method of assay.

Although the organisms grew out more rapidly in culture at 31°C. and 26°C. than they did at 22°C., the organisms died after 21 days at the 2 higher temperatures; while at the lower temperature they continued to multiply for the entire period of observation (42 days), and progressively smaller inocula sufficed to give a positive culture. The method of cultivation which detected the smallest number of organisms proved to be 6 weeks' incubation at 22°C. Under those conditions there was no difference in the sensitivity of the two media tested (N.N.N. and Locke-blood-agar).

The proportion of inadequately treated hamsters giving positive splenic cultures was not significantly different whether the animals were tested 6 weeks, 3 months or 6 months after treatment. However, in the case of antimonials, the fact that the number of organisms in the spleen continues to decrease for as long as 32 days after treatment (4) makes it inadvisable to carry out the test of cure earlier than 6 weeks after treatment. A single negative culture of splenic tissue at that time was estimated to have an 80–85 per cent reliability as a criterion of cure in animals receiving marginal subcurative doses of pentavalent antimonials.

In the light of these data the routine method adopted for the evaluation of cure in experimental leishmaniasis consists of the cultivation on a suitable medium for 6 weeks at 22°C. of material aspirated from the surgically exposed spleen through a 20-gauge needle.

¹ From the Section on Experimental Therapeutics, Laboratory of Infectious Diseases, National Institutes of Health, Bethesda 14, Maryland.

² Presented at the Annual Meeting of the American Society of Tropical Medicine, November 8, 1949, Memphis, Tennessee.