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Effective diagnostic tools are essential in order to combat disease caused by the parasite Entamoeba histolytica. In this study, we compared the commercially available Ridascreen Entamoeba test (R-Biopharm) and the E. histolytica II test (Techlab), and we found that the E. histolytica II test detects E. histolytica infections more accurately.
Entamoeba histolytica, the causative agent of amoebiasis, is currently the only species within the genus Entamoeba known to cause disease in humans (1, 2, 4, 5). Although 90% of E. histolytica infections remain asymptomatic, approximately 50 million cases of amoebic dysentery and 40,000 to 100,000 E. histolytica-induced deaths occur each year (11, 20). Because asymptomatic individuals shed infectious cysts and occasionally develop symptoms over time, both symptomatic and asymptomatic infections necessitate treatment (8). Amoebiasis can be successfully treated with 5-nitroimidazoles and luminal amoebicides, but treatment depends on accurate diagnosis (12, 14).
Microscopy has been used to detect E. histolytica cysts and trophozoites in the stool of infected individuals (6). However, nonpathogenic Entamoeba species, such as E. dispar and E. moshkovskii, confound the issue of diagnostics. E. histolytica and nonpathogenic Entamoeba species are morphologically identical and cannot be differentially detected by microscopy (1, 2, 4). Techniques such as isoenzyme analysis (15, 17) and PCR (7, 14, 18) accurately distinguish between the species, but such techniques are not practical for routine use in developing nations where E. histolytica is prevalent (5, 10, 19).
Enzyme-linked immunosorbent assays (ELISAs) are commonly used in diagnostic laboratories, including those in the developing world. Although there are a few FDA-approved ELISAs for the detection of Entamoeba species, only the Techlab (Blacksburg, VA) E. histolytica II test specifically identifies E. histolytica. The E. histolytica II test has been reported to be more sensitive than the combination of culture and microscopy (9) but only 79% sensitive and 96% specific compared to real-time PCR (14).
The R-Biopharm Ridascreen Entamoeba test (Darmstadt, Germany) detects E. histolytica sensu lato. In one study, the Ridascreen Entamoeba test was found to be 81.8% sensitive and 99.2% specific compared to microscopy (16). However, another study found microscopy 53.8% sensitive and 94% specific compared to the Ridascreen Entamoeba test (3). Although these studies have demonstrated the efficacy of ELISAs as E. histolytica detection methods, no comparison of the commercially available ELISAs exists. In this study we sought to compare the Techlab E. histolytica II test and the R-Biopharm Ridascreen Entamoeba test.
We screened three cultured trophozoite strains and 110 fecal specimens by both tests. E. histolytica HM1:1MSS and Ax 259100 (a patient isolate) were grown axenically in TYI-S-33 (4a), while E. dispar SAW760 was grown xenically in LYI medium (2a). Trophozoites were harvested on ice, and twofold dilution curves ranging from 5.0 × 105 trophozoites per ml to 15 trophozoites per ml were prepared in each kit diluent. The ELISAs were run as recommended by the manufacturers, and each test well received 100 μl of the sample. The trophozoite dilution curves were prepared in duplicate, and within a kit, there was never a discrepancy between replicate samples.
We found the Techlab E. histolytica II test to be more sensitive with respect to the number of trophozoites detected per well. The Techlab test was able to detect as few as 98 HM1:1MSS and 24 Ax 259100 trophozoites per well, while the lowest numbers of trophozoites that the Ridascreen Entamoeba test was able to detect were 195 and 98 per well, respectively. As expected, the Techlab E. histolytica II test was specific for E. histolytica and did not detect any E. dispar trophozoites. The Ridascreen Entamoeba test did detect E. dispar trophozoites. However, the limits of detection for E. dispar were quite high: 25,000 amoebae per well were required to give a positive reaction.
A panel of fecal samples collected from patients with diarrhea at the International Centre for Diarrhoeal Disease Research, Bangladesh (ICDDR,B), was assembled. Upon collection, microscopic examination of a 0.9% saline smear was used to screen samples for ova and parasites, and the samples were then frozen until further use. Samples that tested positive for Entamoeba by microscopy were chosen for the ELISA screen, as were samples positive for other common intestinal organisms, including Ascaris lumbricoides, Giardia spp., Blastocystis hominis, Trichuris trichiura, Cryptosporidium spp., and Entamoeba coli. Microscopy-negative samples were also included in the panel, since microscopy is known to be an insensitive indicator of Entamoeba infection (5, 10).
All samples were diluted in the appropriate kit diluent, and the tests were run and interpreted according to the manufacturers' instructions. Discrepant samples were retested by both ELISAs and analyzed by PCR. The QIAamp DNA stool minikit (Qiagen, Hilden, Germany) was used to extract DNA from the fecal specimens, and PCR for E. histolytica and E. dispar was carried out as previously described (7, 14).
A total of 110 fecal specimens were tested with the R-Biopharm Ridascreen Entamoeba and Techlab E. histolytica II ELISA kits. The E. histolytica II ELISA kit detected 50 E. histolytica-positive samples, while the Ridascreen Entamoeba test identified only 34 positive samples. Twenty-eight samples were positive, and 54 were negative, by both tests. All 22 of the Techlab test-positive, R-Biopharm test-negative samples tested positive for E. histolytica by PCR. Of the six samples that tested positive by the R-Biopharm test but negative by the Techlab test, two contained Entamoeba dispar, two contained E. histolytica, one contained both E. dispar and E. histolytica, and one was negative for both organisms, as determined by PCR. While the PCR-negative sample may represent a false-positive result, it is also possible that the sample contained E. moshkovskii, since the Ridascreen Entamoeba test is not specific for E. histolytica.
The Techlab E. histolytica II ELISA correctly identified approximately 35% (19 of 53) more E. histolytica-positive samples than the Ridascreen Entamoeba test. Additionally, the E. histolytica II test identified exclusively E. histolytica-positive samples, while samples containing E. dispar and possibly E. moshkovskii were detected by the Ridascreen Entamoeba test. Our results indicate that the Techlab E. histolytica II ELISA is not only more specific than the R-Biopharm Ridascreen Entamoeba test but also more sensitive.
Sensitive and specific detection of Entamoeba histolytica infection is required in order to ensure that patients receive the proper treatment. Because extraction of DNA from fecal specimens and PCR remain expensive and require skilled technicians, commercially available ELISAs may currently represent the most practical method for the identification of E. histolytica in fecal samples. While both ELISAs used in this study were relatively quick and easy to perform, the Techlab E. histolytica II ELISA outperformed the R-Biopharm Ridascreen Entamoeba test in our hands.
This work was conducted at the ICDDR,B with the support of NIH grant 5RO1 AI043596-09 through W. A. Petri, Jr., and the University of Virginia. W. A. Petri, Jr., has a patent licensing agreement with Techlab for a diagnostic test for amoebiasis; the royalties from this test accrue to the American Society of Tropical Medicine and Hygiene without benefit to the licensor.
Published ahead of print on 11 June 2008.