Microscopic detection of mixed malarial infections: improvement by saponin hemolysis

The objective of the present study was to determine whether saponin hemolysis could improve microscopic detection of malaria parasites in human blood, since it has been previously reported that the technique has been used to enrich Plasmodium falciparum culture to >/= 90% parasitemia. Blood samples from suspected malaria cases were first examined in routine thick and thin smears under the microscope. The sample [1 ml] was then hemolyzed with 0.015% saponin in saline and centrifuged, the separated pellet was stained with Giemsa stain and examined microscopically, using PCR to confirm species identification. With P. falciparum in vitro culture, the proportions of infected erythrocytes were approximately 0.7-2% before and 65-97% after saponin hemolysis, confirming published reports. In contrast, there was little or no increase in the proportions of intact infected erythrocytes after saponin hemolysis of clinical blood specimens. However, 20-600 hemolyzed parasites were detected per field under the microscope after saponin hemolysis of patients' blood samples that contained only 1-15 parasites per field in conventional thick smears. In addition, more P. falciparum gametocytes were detected after saponin hemolysis. Saponin hemolysis concentrated the parasites in large volumes of blood into a small volume that could be smeared on a slide. This concentration method made it easy to detect malaria parasites and reduced the time needed for microscopy. In the present study, the method was comparable to PCR for the identification of P. vivax and P. falciparum mixed infections

Gender identification of highly degraded DNA extracted from human compact bone samples

The present work revealed a sensitive and reliable method for sex typing of degraded DNA extracted from bones subjected to different environmental conditions, based on amplification of the single- copy X- Y homologous amelogenin gene using nested PCR technology. The success of retrieval of amplifiable amelogenin was related to the storage conditions, bones exposed to open air were found to provide sufficiently preserved DNA, followed by sand buried bones then sea water submerged samples. The success rate of the amelogenin amplification was 88.9%, 66.7% and 46.7%, respectively. Moreover, the method of DNA extraction with high salt, followed by phenol- chloroform gave the best results. This test enabled gender identification from as low as 3 ng DNA, suggesting that even a small fragment of a bone was sufficient to carry out the procedure of isolation of DNA for sex typing