A novel nested multiplex polymerase chain reaction (PCR) assay for differential detection of Entamoeba histolytica, E. moshkovskii and E. dispar DNA in stool samples

Abstract

Background: E. histolytica, a pathogenic amoeba, is indistinguishable in its cyst and trophozoite stages from those of non-pathogenic E. moshkovskii and E. dispar by light microscopy. We have developed a nested multiplex PCR targeting a 16S-like rRNA gene for differential detection of all the three morphologically similar forms of E. histolytica, E. moshkovskii and E. dispar simultaneously in stool samples.

Results: The species specific product size for E. histolytica, E. moshkovskii and E. dispar was 439, 553 and 174 bp respectively, which was clearly different for all the three Entamoeba species. The nested multiplex PCR showed a sensitivity of 94% and specificity of 100% for the demonstration of E. histolytica, E. moshkovskii and E. dispar DNA in stool samples. The PCR was positive for E. histolytica, E. moshkovskii and E. dispar in a total of 190 out of 202 stool specimens (94% sensitive) that were positive for E. histolytica/E. dispar/E. moshkovskii by examination of stool by microscopy and/or culture. All the 35 negative control stool samples that were negative for E. histolytica/E. dispar/E. moshkovskii by microscopy and culture were also found negative by the nested multiplex PCR (100% specific). The result from the study shows that only 34.6% of the patient stool samples that were positive for E. histolytica/E. dispar/E. moshkovskii by examination of stool by microscopy and/or culture, were actually positive for pathogenic E. histolytica and the remaining majority of the stool samples were positive for non-pathogenic E. dispar or E. moshkovskii as demonstrated by the use of nested multiplex PCR.

Conclusion: The present study reports a new nested multiplex PCR strategy for species specific detection and differentiation of E. histolytica, E. dispar and E. moshkovskii DNA in stool specimens. The test is highly specific, sensitive and also rapid, providing the results within 12 hours of receiving stool specimens.

Figure 1

Sensitivity of PCR for detection of minimum number of E. moshkovskii (EM), E. histolytica (EH) and E. dispar (ED) cells. 0.05 gm of negative control stool specimen seeded with serially diluted Entamoeba cells corresponding to 10⁶ cells (lane 1), 10⁵ cells (lane 2), 10⁴ cells (lane 3), 10³ cells (lane 4), 10² cells (lane 5), and 10 cells (lane 6) were subjected to DNA extraction followed by PCR amplification. Amplified products were analyzed by agarose gel electrophoresis. The sizes of the amplification products are indicated on the left (in base pairs). Lanes 7, negative control (PCR without DNA)

Figure 2

Detection of E. histolytica (A), E. dispar (B) and E. moshkovskii (C) in mixed cell lysates. To 1000 cells of E. dispar and E. moshkovskii (A) or E. histolytica and E. moshkovskii or (B), E. histolytica and E. dispar (C), 1000 cells (lane 1), 100 cells (lane 2), 10 cell (lane 3), 1 cell (lane 4), 0.1 cell (lane 5), 0.01 cell (lane 6) and 0.001 cell (lane 7) of the other species were added. Amplification was done by nested multiplex PCR.

Figure 3

Differential detection of E. histolytica, E. moshkovskii and E. dispar by nested multiplex PCR on stool samples. The E. moshkovskii (EM), E. histolytica (EH) and E. dispar (ED) bands are 553, 439 and 174 bp, respectively. Lane-1, E. moshkovskii (mono infection); Lane-2, E. moshkovskii, E. histolytica and E. dispar (mixed infection); Lane-3, E. histolytica and E. dispar (mixed infection); Lane-4 E. moshkovskii and E. histolytica (mixed infection); Lane-5, E. moshkovskii and E. dispar (mixed infection); Lane-6 E. histolytica (mono infection), Lane-7 E. dispar (mono infection); Lane-8, 100 bp DNA ladder (Bangalore genei, Bangalore).