Comparison of multiplex-PCR and antigen detection for differential diagnosis of Entamoeba histolytica

Abstract

Amebiasis is an infection caused by Entamoeba histolytica. However, differentiation between E. histolytica and Entamoeba dispar, which are morphologically identical species, is essential for treatment decision, precaution of the invasive disease and public health. The purpose of the present study was to evaluate a Multiplex -PCR for detection and differentiation of E. histolytica from E. dispar from fresh stool samples in comparison with the coproantigen commercial ELISA. Microscopic examination of stools using the Coprotest method, detection of stool antigen by enzyme-linked immunosorbent assay kit and a home made Multiplex-PCR, were used for the diagnosis of amoebiasis infection. Analysis of the 127 stools samples by microscopy examination demonstrated that only 27 (21%) samples were positive for E. histolytica/E. dispar complex. Among these stool samples, 11 were positive by Multiplex-PCR, with nine presenting the diagnostic fragment characteristic of E. dispar (96 bp) and two presenting diagnostic fragment of E. histolytica (132 bp). Among negative samples detected by microscopic examination, three positive samples for E. dispar and one positive for E. histolytica by Multiplex-PCR was observed. This denotes a low sensibility of microscopic examination when a single stool sample is analyzed. Assay for detection of E. histolytica antigen was concordant with multiplex-PCR in relation to E. histolytica. Statistical analysis comparing the sensibility tests was not done because of the low number of E. histolytica cases. The results demonstrate the importance of the specific techniques use for the differentiation between E. histolytica and E. dispar.

Identification of Entamoeba histolytica and E. dispar infection in Maceió, Alagoas State, northeast Brazil

“…In general, there is a higher number of E. dispar cases in relation to the E. histolytica infections, as reported in other studies, including in Brazil, as well as the occurrence of mixed infections with E. histolytica and E. dispar [18][19][20][21][22].…”

Differential identification of Entamoeba spp. based on the analysis of 18S rRNA

“…However, such approaches are not available for the differentiation of the other Entamoeba spp. DNA-based approaches are very convenient for this type of work, since these methods can be multiplexed to allow identification of multiple pathogens simultaneously (Dunbar 2006;Ali et al 2003;Verweij et al 2003a;Santos et al 2007;Ben et al 2008). Among them, several polymerase chain reaction (PCR)-based tests have been developed for the differentiation of E. histolytica, E. dispar, and more recently for E. moshkovskii in stools.…”

Frequency of amoebiasis and other intestinal parasitoses in a settlement in Ilhéus City, State of Bahia, Brazil

“…However, the prevalence of Entamoeba histolytica in the country should be reassessed 4 because the existing epidemiological data are based on the microscopic examination of feces, which does not provide suitably accurate results. In recent years, diagnoses of amoebiasis have focused on the identification of the parasitic antigen in the feces and on the detection of Entamoeba dispar and E. histolytica deoxyribonucleic acid (DNA) by polymerase chain reaction (PCR) 5,6 . Entamoeba hartmanni has smaller dimensions and can thus be differentiated from the E. histolytica/E.…”

“…dispar complex were stained with iron hematoxylin. Multiplex PCR specific for the detection and differentiation of E. histolytica and E. dispar 6 and an E. histolytica stool antigen test (E. histolytica II enzyme-linked immunosorbent assay) test kit, TECHLAB Inc., Blacksburg, VA, USA) were employed to differentiate the E. histolytica/E. dispar The overall frequency of intestinal parasitoses was 65%, including 37 (70%) of 53 analyzed males and 26 (59%) of 44 analyzed females.…”