Polymerase chain reaction-solution hybridization enzyme-linked immunoassay (PCR-shela) for the differential diagnosis of pathogenic and non-pathogenic Entamoeba histolytica

Aguirre, Aura; Warhurst, David C.; Guhl, Felipe; Frame, I. A.

doi:10.1016/0035-9203(95)90488-3

Cited by 34 publications

(22 citation statements)

References 5 publications

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“…17,30 This procedure is apparently 100% specific but not 100% sensitive when applied to stored fecal samples (Shire and Ackers, unpublished observations). Thus, whereas a positive result confirms the diagnosis, a negative one does not exclude it; therefore, an adequate sample storage and conservation procedure is of great importance.…”

Section: Discussionmentioning

confidence: 99%

“…in feces. 17 Usually, the identification of specific antibodies against antigens of E. histolytica does not correlate with resistance to the infection or with protective immunity. 18 However, detection of seric antibodies is a useful procedure in the diagnosis of invasive extra-intestinal amoebiasis, but not in the intestinal forms, where it is not feasible to distinguish asymptomatic carriers from patients with amoebic dysentery, especially in endemic areas where antibody titer is not enough to diagnose acute intestinal amoebiasis.…”

Section: Introductionmentioning

confidence: 99%

“…The molecular diagnosis was made by Colorimetric PCR as described. 17,30 The DNA from E. histolytica HM1-IMSS and from E. dispar clone 53 31 was used as a positive control.…”

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confidence: 99%

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Differentiation of entamoeba histolytica/entamoeba dispar by PCR and their correlation with humoral and cellular immunity in individuals with clinical variants of amoebiasis.

Sánchez-Guillén¹,

Pérez-Fuentes

Salgado-Rosas

et al. 2002

The American Journal of Tropical Medicine and Hygiene

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Abstract. To correlate a particular state of immunity with Entamoeba spp., we used colorimetric PCR to differentiate E. histolytica from E. dispar in individuals with amoebiasis and to associate its presence with the clinical profile, including humoral and cellular immune responses to E. histolytica. Our results showed high levels of antibody in acute amoebiasis and elevation of IL-4 production, a cytokine related to Th2 profile, associated with E. histolytica. In chronic amoebiasis, even with anti-E. histolytica seropositivity, intestinal symptoms were associated with E. dispar in all the cases, without differences in level of antibodies, BTI, CD4+/CD8+ ratio, INF-␥, and IL-4. Among asymptomatic carriers, E. dispar was more frequently found; however, identification of E. histolytica in two asymptomatic carriers associated with high levels of INF-␥, a cytokine related to Th1 profile, demonstrate the importance of making specific diagnosis of Entamoeba spp., to establish the clinical and epidemiological behavior in both intestinal and extra-intestinal amoebiasis.

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Section: Discussionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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Differentiation of entamoeba histolytica/entamoeba dispar by PCR and their correlation with humoral and cellular immunity in individuals with clinical variants of amoebiasis.

Sánchez-Guillén¹,

Pérez-Fuentes

Salgado-Rosas

et al. 2002

The American Journal of Tropical Medicine and Hygiene

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“…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.…”

Section: Amebiasis Is An Important Parasitic Disease In Humans (18)mentioning

confidence: 99%

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

2003

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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...

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“…In any event, the results obtained during the study of the Meadowlark Cemetery sediment samples should encourage others to use various immunological techniques, or immunological and molecular techniques together. Such techniques might include immunofluorescence or polymerase chain reaction-solution hybridation enzyme-linked immunoassay (Aguirre et al 1995(Aguirre et al , 1997) that could be more sensitive for studying the evolution of the parasites genome.…”

Section: Resultsmentioning

confidence: 99%

Parasitism in Kansas in the 1800s: a glimpse to the past through the analysis of grave sediments from Meadowlark cemetery

Bailly

Gonçalves

Lefèvre

et al. 2006

Mem. Inst. Oswaldo Cruz

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During the excavations of the XIX century Meadowlark cemetery (Manhattan, Kansas, US) Scientists have been using different techniques to look for parasites in ancient material (Reinhard et al. 1987, Araujo et al. 1998). Coprolites or desiccated feces, or fecal remains mixed with soil sediment are commonly found in archaeological excavations. The parasitological analysis of such material yields rich information about paleopathologies, human migrations, cultural changes, dietary habits, paleoclimates, parasite dispersion, and host migrations throughout space and time (Reinhard et al 2001, Gonçalves et al. 2003. MATERIALS AND METHODSThe analyzed sediments were collected during the excavation of the Meadowlark Cemetery, on the grounds of Meadowlark Hills Retirement Community in Manhattan, Kansas. This cemetery was used during the period ca. 1860 to 1900. It was excavated during the summer and early fall of 2004 to make way for construction of new residences on the retirement community property. A total of 17 graves were revealed. Thirteen of these graves contained skeletons. Sediment samples from the abdominal region of five skeletons were studied: MC F9, F10, F12, F13, and F17 (Table).The material was submitted to the standard paleoparasitological examination using an optical microscope to search for helminth eggs. A commercially available enzyme-linked immunosorbent assay (ELISA) with monoclonal antibodies was used to test for antigen from Entamoeba histolytica (Haque et al. 1995(Haque et al. , 2000 and Giardia duodenalis (Aldeen et al. 1998, Boone et al. 1999, two common intestinal protozoan parasites.For the standard paleoparasitological examination, the samples were rehydrated for a week in 0.5% trisodic phosphate and 5% glycerinated solution. The material was then crushed in a mortar. The suspension was submitted to an ultrasound device for 1 min at 50°C, on a frequency of 35 kHz, and the suspension strained through 315-µm, 160-µm, 50-µm, and 25-µm meshes. A portion of each sample was used for microscopic examination. The material was placed on a slide that was covered with a coverslip and examined for evidence of parasites and food remains. Twenty slides of each sample were examined at magnification of ×100 and ×400 (Bouchet et al. 2001).For the ELISA tests, the E. histolytica II and the Giardia II assay (TechLab, Blackburg, VA, US) were used. Sample preparation was quite different to provide physicochemical parameter modifications, especially due to formalin (Hould 1984, Exbrayat 2000. The samples were rehydrated for one week in ultra-pure water obtained from a purification system manufactured by Millipore ® (Direct-Q TM 5). Samples were preserved in the freezer (2-4°C) during the rehydration phase to avoid bacteria and fungi development. Manufacturer's directions were followed and results were interpreted by visual inspection and using a spectrophotometer. A test sample was considered positive if it had an obvious yellow color when compared to the negative control well. A test sample was considered negativ...

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Polymerase chain reaction-solution hybridization enzyme-linked immunoassay (PCR-shela) for the differential diagnosis of pathogenic and non-pathogenic Entamoeba histolytica

Cited by 34 publications

References 5 publications

Differentiation of entamoeba histolytica/entamoeba dispar by PCR and their correlation with humoral and cellular immunity in individuals with clinical variants of amoebiasis.

Differentiation of entamoeba histolytica/entamoeba dispar by PCR and their correlation with humoral and cellular immunity in individuals with clinical variants of amoebiasis.

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

Parasitism in Kansas in the 1800s: a glimpse to the past through the analysis of grave sediments from Meadowlark cemetery

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