Detection and differentiation of Entamoeba histolytica and Entamoeba dispar in clinical samples through PCR-denaturing gradient gel electrophoresis

Amebiasis is one of the twenty major causes of disease in Mexico; however, the diagnosis is difficult due to limitations of conventional microscopy-based techniques. In this study, we analyzed stool samples using polymerase chain reaction-denaturing gradient gel electrophoresis (PCR-DGGE) to differentiate between Entamoeba histolytica (pathogenic) and E. dispar (non-pathogenic). The target for the PCR amplification was a small region (228 bp) of the adh112 gene selected to increase the sensitivity of the test. The study involved 62 stool samples that were collected from individuals with complaints of gastrointestinal discomfort. Of the 62 samples, 10 (16.1%) were positive for E. histolytica while 52 (83.9%) were negative. No sample was positive for E. dispar. These results were validated by nested PCR-RFLP (restriction fragment length polymorphism) and suggest that PCR-DGGE is a promising tool to differentiate among Entamoeba infections, contributing to determine the specific treatment for patients infected with E. histolytica, and therefore, avoiding unnecessary treatment of patients infected with the non-pathogenic E. dispar.

Frequency and molecular characterisation of Entamoeba histolytica, Entamoeba dispar, Entamoeba moshkovskii, and Entamoeba hartmanni in the context of water scarcity in northeastern Brazil

This study aimed to estimate the frequency, associated factors, and molecular characterisation of Entamoeba histolytica, Entamoeba dispar, Entamoeba moshkovskii, andEntamoeba hartmanni infections. We performed a survey (n = 213 subjects) to obtain parasitological, sanitation, and sociodemographic data. Faecal samples were processed through flotation and centrifugation methods.E. histolytica, E. dispar, E. moshkovskii, and E. hartmanni were identified by nested-polymerase chain reaction (PCR). The overall prevalence of infection was 22/213 (10.3%). The infection rate among subjects who drink rainwater collected from roofs in tanks was higher than the rate in subjects who drink desalinated water pumped from wells; similarly, the infection rate among subjects who practice open defecation was significantly higher than that of subjects with latrines. Out of the 22 samples positive for morphologically indistinguishableEntamoeba species, the differentiation by PCR was successful for 21. The species distribution was as follows: 57.1% to E. dispar, 23.8% to E. histolytica, 14.3% toE. histolytica and E. dispar, and 4.8% E. dispar and E. hartmanni. These data suggest a high prevalence of asymptomatic infection by the group of morphologically indistinguishable Entamoeba histolytica/dispar/moshkovskiicomplex and E. hartmanni species. In this context of water scarcity, the sanitary and socioenvironmental characteristics of the region appear to favour transmission.

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

Introduction: This study evaluated the frequency of intestinal parasites, emphasizing the identification and differentiation of Entamoeba spp. Methods: Multiplex polymerase chain reaction (PCR), coproantigen tests and morphometric analysis were performed for Entamoeba spp. differentiation. Results: The overall frequency of intestinal parasites was 65%. Entamoeba histolytica was detected by the coproantigen test, and the PCR showed that Entamoeba dispar predominated in the population. In contrast, morphometric analysis was important for identifying Entamoeba hartmanni. Conclusions: It is possible to identify the causative agent of amoebiasis and to differentiate this agent from other species by combining techniques. .

Diferenciación del complejo Entamoeba histolytica/Entamoeba dispar mediante Gal/GalNAc-lectina y PCR en aislamientos colombianos / Differentiation of entamoeba histolytica from entamoeba dispar using Gal/GalNAc-lectin and polymerase chain reaction

Background: Entamoeba histolytica and Entamoeba dispar are morphologically identical. However, the former is highly pathogenic and the latter is not. Aim: To differentiate Entamoeba histolytica from Entamoeba dispar through ELISA and PCR techniques in Colombian isolates from feces. Material and Methods: Descriptive study of Colombian fecal samples from 53 males and 47 women, that were positive for the complex E. histolytica/E. dispar on light microscopy. Positive samples were cultured on Robinson medium to isolate trophozoites. The presence of specific Gal/ GalNAc-lectin was determined by ELISA and polymerase chain reaction in genomic DNA, using the combination of three nucleotides that recognize a variable region of 16S small subunit ribosomal RNA, generating a 166 base pair (bp) product for E. histolytica and 752 pb product for E. dispar. Results: After verification, only eight of the 100 samples were positive for the complex E. histolytica/E. dispar and were cultivated. Isolates were obtained in six cultures, one corresponded to E. histolytica and six to E. dispar. Conclusions: The presence of E. histolytica/E. dispar complex was largely overestimated with light microscopy. In the few samples where isolates were obtained, the technique described differentiated between both strains.

Validation and utilization of PCR for differential diagnosis and prevalence determination of Entamoeba histolytica/Entamoeba dispar in Salvador City, Brazil

Amoebiasis is an infection caused by Entamoeba histolytica and is a potential health risk in countries in which health barriers are inappropriate. Since the discovery of Entamoeba dispar, the prevalence of amoebiasis has been modified. OBJECTIVE: This study has standardized the PCR technique applied for the diagnosis of different species of the E. histolytica/E. dispar complex and has evaluated the prevalence of infection among patients attending private and public clinical laboratories in Salvador City, Bahia State, Brazil. RESULTS: Analysis of 52,704 stool samples by microscopic examination demonstrated that 1,788 (3.4 percent) were positive for the E. histolytica/E. dispar complex and infection occurred more often in samples originated from public clinical laboratories (5.0 percent) than those that came from private laboratories (3.2 percent). PCR performed in approximately 15 percent (262) E. histolytica/E. dispar complex positive samples, randomly chosen, amplified 227 samples (86.6 percent), all of them positive for E. dispar. The non-amplified 35 samples (13.4 percent) were also negative for E. histolytica-specific galactose adhesin. Moreover, to exclude a probable infection caused by E. hartmanni, morphometric analysis demonstrated that non-amplified samples had cyst sizes comparable to E. histolytica/E. dispar (>10 µm). CONCLUSION: The absence of amplification of these samples indicates the presence of PCR inhibitors in the stool samples or the presence of DNA from Entamoeba species other than E. dispar, E. histolytica or E. hartmanni.

Detección y diferenciación de Entamoeba histolytica y Entamoeba dispar mediante reacción en cadena de la polimerasa en individuos de una comunidad del Estado Zulia, Venezuela / Detection and differentiation of Entamoeba histolytica and Entamoeba dispar by polymerase chain reaction in a community in Zulia State, Venezuela

Differential identification of Entamoeba histolytica and Entamoeba dispar is essential for both appropriate patient treatment and epidemiological purposes. To determine the prevalence of these amoeba infections in Santa Rosa de Agua (Maracaibo, Zulia State, Venezuela), a PCR assay using specific primers for each species was standardized and applied. 204 stool samples were analyzed through direct microscopic examination with SSF (0.85 percent) and lugol, formol-ether concentration, and PCR. Under direct microscopy, 42 individuals (20.58 percent) presented the E. histolytica/E. dispar complex. Meanwhile PCR showed 47 positive cases for these amoebas: 22 E. histolytica (10.78 percent), 16 E. dispar (7.84 percent), and 9 (4.41 percent) mixed infections. There was no significant difference in the presence of E. histolytica and/or E. dispar according to either gender or age. There were no cases of these amoebas in children under 2 years of age. Observed frequency of E. histolytica (31/204) shows the endemic nature of amoeba infection in this community.

La identificación diferencial de Entamoeba histolytica y Entamoeba dispar es esencial para un tratamiento adecuado del paciente y con fines epidemiológicos. Para determinar la prevalencia de E. histolytica y E. dispar se estandarizó y aplicó un ensayo de PCR, utilizando oligonucleótidos específicos para cada especie. 204 muestras de heces de individuos de la comunidad de Santa Rosa de Agua (Municipio Maracaibo, Estado Zulia, Venezuela), fueron analizadas a través del examen directo con SSF (0,85 por ciento) y lugol, concentrado de formol-éter y PCR. Al examen microscópico, 42 individuos (20,58 por ciento) presentaron formas evolutivas del complejo E. histolytica/E. dispar; mientras que la técnica de PCR evidenció un total de 47 casos positivos a estas amibas; de los cuales 22 eran portadores de E. histolytica (10,78 por ciento), 16 (7,84 por ciento) de E. dispar y 9 (4,41 por ciento) presentaron infección mixta. No hubo diferencia significativa al relacionar las variables sexo y presencia de E. histolytica y/o E. dispar, ni con los grupos etarios. No existieron casos de estas amibas, en los menores de 2 años. La frecuencia observada de E. histolytica (31/204), demuestra el carácter endémico de la amibiasis en esta comunidad.

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

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 percent) 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.