Detection and Differentiation of Entamoeba histolytica and Entamoeba dispar Isolates in Clinical Samples by PCR and Enzyme-Linked Immunosorbent Assay

Abstract

Differential diagnosis of Entamoeba histolytica (pathogenic) and Entamoeba dispar (nonpathogenic), which are two morphologically identical species of amebae, is essential both for treatment decision and public health knowledge. The study reported here was designed to choose a reference differentiation technique. Stool samples (n ‫؍‬ 95) were tested by microscopy, TechLab enzyme-linked immunosorbent assays (ELISAs), and an in-house PCR. The target for the PCR amplification was a small region (135 bp) of the SSU rRNA selected to increase the sensitivity of the test. Sixty-eight specimens tested positive by PCR: 2 for E. histolytica and 66 for E. dispar. For detection of E. dispar, ELISA performance was lower than that of microscopy in this reference context, while PCR was much more sensitive than microscopy. Given the low proportion of E. histolytica cases, test performance for this species is difficult to assess. However, for differentiation, PCR performed well on simulated samples, while ELISA gave a discordant result for one of the two samples PCR positive for E. histolytica during the study. This report also confirms that E. dispar infection is significantly higher among travelers and underlines the possibility of acquiring E. histolytica infection in regions that are not areas of endemicity. Because of its lower sensitivity, the interest of ELISA for Entamoeba detection and differentiation in stools seems questionable in nontropical regions. On the other hand, results suggest that PCR should be useful as a reference test for sensitive differentiation of both species and to contribute to physicians' decision in treatment of E. histolytica-or E. dispar-infected patients. Amebiasis is an important parasitic disease in humans (18).Entamoeba histolytica and Entamoeba dispar parasitize approximately 10% of the world population, of which 90% are asymptomatic infections. It is estimated, however, that amebiasis causes up to 110,000 deaths a year (15). While the infectious agent was discovered in 1875 by Fedor A. Lösch and the distinction between E. dispar and E. histolytica was first suspected in 1925 (3), the evidence for the dichotomy in two different species, pathogenic (E. histolytica) and nonpathogenic (E. dispar), is relatively recent (5). However, E. dispar and E. histolytica are morphologically indistinguishable from one another. Isoenzyme analysis is considered the "gold standard" for differentiating E. histolytica and E. dispar, but this method is not currently available and not readily usable for routine diagnosis (16). More recently, several studies have been devoted to the development of new techniques either based on monoclonal antibodies (8, 9, 10, 20, 21) or molecular biology methods (1,2,4,6,19,22,23) to successfully distinguish the two species in human feces. Reliable distinction would have a medical impact as until now, both infections are usually treated, whereas only approximately 10% (pathogenic infections) need to be treated. This proportion drops to much lower levels in developed countries, wh...

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

“…Several PCR assays designed to differentiate E. histolytica for E. dispar have been described [5,6,21,[22][23][24][25]32,33]. Most of them targeted either the small subunit ribosomal RNA gene or specific episomal repeats species.…”

“…This feature has been observed among patients with dysentery. Some investigators suggest that this classical feature has long been considered the definitive diagnostic criterion for E. histolytica [1,6,11,14]. However, it is rarely observed in chronic amoebic infections.…”

“…It has been related that majority of individuals infected are asymptomatic [4][5][6][7][8]. These individuals are reservoir of infection and represent the most neglected category of infected subjects, what might interfere in an epidemiological study and in control of this infectious.…”

“…The coproantigen ELISA technique has been suggested to be used in routine diagnosis procedure and epidemiologic studies. However, a comparative study on the use of the ELISA and PCR for detection of E. histolytica indicated that PCR was more sensitive [6,20]. In reference laboratories, PCR is the method of choice for differentiation between the pathogenic specie (E. histolytica) from the non-pathogenic (E. dispar).…”

“…In reference laboratories, PCR is the method of choice for differentiation between the pathogenic specie (E. histolytica) from the non-pathogenic (E. dispar). Many investigations have reported successful application of PCR to the diagnosis of amoebiasis as a tool for final confirmatory identification of intestinal amoebiasis [5,6,[20][21][22][23][24][25][26].…”