Improved diagnosis of Trichuris trichiura by using a bead-beating procedure on ethanol preserved stool samples prior to DNA isolation and the performance of multiplex real-time PCR for intestinal parasites

摘要

For the majority of intestinal parasites, real-time PCR-based diagnosis outperforms microscopy. However, the data for Trichuris trichiura have been less convincing and most comparative studies have been performed in populations with low prevalence. This study aims to improve detection of T. trichuria DNA in human stool by evaluating four sample preparation methods. Faecal samples (n = 60) were collected at Flores island, Indonesia and examined by microscopy. Aliquots were taken and a bead-beating procedure was used both on directly frozen stool and on material preserved with 96% ethanol. PCR on frozen samples showed 40% to be positive for T. trichiura, compared with 45% positive by microscopy. The percentage positive increased when using ethanol preservation (45.0%), bead-beating (51.7%) and a combination (55.0%) and all three methods showed significantly higher DNA loads. The various procedures had a less pronounced effect on the PCR results of nine other parasite targets tested. Most prevalent were Ascaris lumbricoides (approximate to 60%), Necator americanus (approximate to 60%), Dientamoeba fragilis (approximate to 50%) and Giardia lamblia(approximate to 12%). To validate the practicality of the procedure, bead-beating was applied in a population-based survey testing 910 stool samples. Findings confirmed bead-beating before DNA extraction to be a highly efficient procedure for the detection of T. trichiura DNA in stool.