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

Abstract

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.

Keywords: Trichuris trichiura; bead-beating; intestinal parasite; real-time PCR; sample preparation.

Fig. 1.

Flow-chart of the collection, preparations and measurements of 60 stool samples. Each preparation procedure is labelled as: 1a=C_PCR: PCR from directly frozen sample; 2a=E_PCR: PCR from ethanol preserved samples; 1b=B_PCR: PCR from bead-beating supplemented on frozen sample; 2b=E_B_PCR: PCR from bead-beating supplemented on ethanol-preserved samples. Real-time PCR detection: Panel I=ST (targetting Schistosoma sp. and T. trichiura); Panel II=ANAS (targetting A. duodenale, N. americanus, A. lumbricoides and S. stercoralis); Panel III=HDGC (targetting E. histolytica, D. fragilis, G. lamblia and Cryptosporidium spp.).

Fig. 2.

DNA load distribution of five most prominent intestinal parasites using four different preparation procedures on 60 stool samples. PCR results following the sample preparation procedure: C_PCR = directly frozen sample; E_PCR = ethanol-preserved sample; B_PCR = bead-beating supplemented on frozen sample; E_B_PCR = bead-beating supplemented on ethanol-preserved samples. Tt, T. trichiura; Na, N. americanus; Al, A. lumbricoides; Gl, G. lamblia; Df, D. fragilis.

Fig. 3.

Association between egg output (Log EPG) and DNA load (Ct value) for T. trichiura (A–D) and A. lumbricoide (E–H). PCR results following the sample preparation procedure: C_PCR = directly frozen sample; E_PCR = ethanol-preserved sample; B_PCR = bead-beating supplemented on frozen sample; E_B_PCR = bead-beating supplemented on ethanol-preserved samples. Samples negative for both microscopy and real-time PCR were excluded in the statistical analysis.

Fig. 4.

Prevalence and intensity of T. trichiura detected by PCR and Kato smear (KS) in 455 individuals before and 1 year after intense albendazole treatment. Ct values generated from real-time PCR were divided into three groups: high DNA load= Ct < 30, moderate DNA Load= 30 ⩽ Ct < 35 and low DNA load= 35 ⩽ Ct < 50. EPG is calculated from Kato smear detection and divided into three categories based on WHO criteria: heavy−=⩾10,000, moderate−= 1000–9999, and light-infection= 1–999.