Detection and differentiation of Entamoeba histolytica and Entamoeba dispar in clinical samples through PCR-denaturing gradient gel electrophoresis

Abstract

Amebiasis is one of the twenty major causes of disease in Mexico; however, the diagnosis is difficult due to limitations of conventional microscopy-based techniques. In this study, we analyzed stool samples using polymerase chain reaction-denaturing gradient gel electrophoresis (PCR-DGGE) to differentiate between Entamoeba histolytica (pathogenic) and E. dispar (non-pathogenic). The target for the PCR amplification was a small region (228 bp) of the adh112 gene selected to increase the sensitivity of the test. The study involved 62 stool samples that were collected from individuals with complaints of gastrointestinal discomfort. Of the 62 samples, 10 (16.1%) were positive for E. histolytica while 52 (83.9%) were negative. No sample was positive for E. dispar. These results were validated by nested PCR-RFLP (restriction fragment length polymorphism) and suggest that PCR-DGGE is a promising tool to differentiate among Entamoeba infections, contributing to determine the specific treatment for patients infected with E. histolytica, and therefore, avoiding unnecessary treatment of patients infected with the non-pathogenic E. dispar.

Figure 1. Alignment sequence between the adh112 gene fragments from E. histolytica and E. dispar. Five differences between sequences are highlighted.

Figure 2. Stained agarose gel with products amplified by PCR with designed primers from samples containing E. histolytica or E. dispar. Lane 1, molecular weight marker; lanes 2–4, clinical samples; lane 5, DNA positive control for E. histolytica (c+eh); lane 6, DNA positive control for E. dispar (c+ed); and lane 7, negative control (human DNA from whole blood).

Figure 3. Denaturing gradient gel electrophoresis analysis of stool samples for the identification of E. histolytica and E. dispar. Lane 1, positive control of E. dispar; lane 2, positive control of E. histolytica; lanes 3–6, clinical samples; lane 7, negative control.

Figure 4. Nested PCR-RFLP (restriction fragment length polymorphism) of SSU rRNA gene for the identification of E. histolytica and E. dispar. Lane 1, molecular weight marker; lanes 2–5, DraI digested PCR products; lane 6, DNA positive control for E. histolytica (c+eh); lane 7–10, Sau96I digested PCR products; lane 11, DNA positive control for E. dispar (c+ed); and lane 12, negative control.