ABSTRACT
Background &
objectives:
Amoebiasis is a common
parasitic infection caused by
Entamoeba histolytica and
amoebic liver abscess (ALA) is the most common extraintestinal manifestation of
amoebiasis. The aim of this study was to standardise
real-time PCR assays (Taqman and SYBR Green) to detect E. histolytica from
liver abscess pus and stool samples and compare its results with nested-
multiplex PCR.
Methods:
Liver abscess pus specimens were subjected to
DNA extraction. The extracted
DNA samples were subjected to amplification by nested-
multiplex PCR, Taqman (
18S rRNA) and SYBR Green
real-time PCR (16S-like rRNA assays to detect E. histolytica/E. dispar/E. moshkovskii). The amplification products were further confirmed by
DNA sequence analysis. Receiver operator characteristic (ROC) curve
analysis was done for nested-multiplex and SYBR Green
real-time PCR and the area under the curve was calculated for evaluating the accuracy of the tests to dignose ALA.
Results:
In all, 17, 19 and 25
liver abscess samples were positive for E. histolytica by nested-
multiplex PCR, SYBR Green and Taqman
real-time PCR assays, respectively. Significant differences in
detection of E. histolytica were noted in the
real-time PCR assays evaluated (P<0.0001). The nested-
multiplex PCR, SYBR Green
real-time PCR and Taqman
real-time PCR evaluated showed a positivity rate of 34, 38 and 50 per cent, respectively. Based on
ROC curve analysis (considering Taqman
real-time PCR as the
gold standard), it was observed that SYBR Green
real-time PCR was better than conventional nested-
multiplex PCR for the
diagnosis of ALA. Interpretation &
conclusions:
Taqman
real-time PCR targeting the
18S rRNA had the highest positivity rate evaluated in this study. Both nested multiplex and SYBR Green
real-time PCR assays utilized were evaluated to give accurate results.
Real-time PCR assays can be used as the
gold standard in rapid and reliable
diagnosis, and appropriate management of
amoebiasis, replacing the conventional molecular
methods.