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

• Published in: Parasitology 2017-06, Vol.144 (7), p.965-974
• Main Authors: KAISAR, MARIA M. M., BRIENEN, ERIC A. T., DJUARDI, YENNY, SARTONO, ERLIYANI, YAZDANBAKHSH, MARIA, VERWEIJ, JACO J., SUPALI, TANIAWATI, VAN LIESHOUT, LISETTE
• Format: Article
• Language: English
• Subjects: Adult; Animals; Child, Preschool; Comparative studies; Deoxyribonucleic acid; Diagnosis; DNA; Ethanol; Ethanol - chemistry; Feces; Feces - parasitology; Female; Giardia; Humans; Indonesia - epidemiology; Infant; Infections; Intestinal parasites; Intestine; Laboratories; Loads (forces); Medicine; Microscopy; Middle Aged; Multiplex Polymerase Chain Reaction; Multiplexing; Parasites; Parasitology; Polymerase chain reaction; Preservation; Prevalence; Real time; Real-Time Polymerase Chain Reaction; Sample preparation; Sampling methods; Specimen Handling; Studies; Trichuriasis - diagnosis; Trichuriasis - epidemiology; Trichuriasis - parasitology; Trichuris - isolation & purification; Young Adult
• ISSN: 0031-1820; 1469-8161
• DOI: 10.1017/S0031182017000129

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 (≈60%), Necator americanus (≈60%), Dientamoeba fragilis (≈50%) and Giardia lamblia (≈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.