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.

Laboratory diagnosis of amebiasis in a sample of students from southeastern Brazil and a comparison of microscopy with enzyme-linked immunosorbent assay for screening of infections with Entamoeba sp.

Introduction: Epidemiological studies on amebiasis have been reassessed since Entamoeba histolytica and E. dispar were first recognized as distinct species. Because the morphological similarity of these species renders microscopic diagnosis unreliable, additional tools are required to discriminate between Entamoeba species. The objectives of our study were to compare microscopy with ELISA kit (IVD®) results, to diagnose E. histolytica infection, and to determine the prevalence of amebiasis in a sample of students from southeastern Brazil. Methods: In this study, diagnosis was based on microscopy due to its capacity for revealing potential cysts/trophozoites and on two commercial kits for antigen detection in stool samples. Results: For 1,403 samples collected from students aged 6 to 14 years who were living in Divinópolis, Minas Gerais, Brazil, microscopy underestimated the number of individuals infected with E. histolytica/E. dispar (5.7% prevalence) compared with the ELISA kit (IVD®)-based diagnoses (15.7% for E. histolytica/E. dispar). A comparison of the ELISA (IVD®) and light microscopy results returned a 20% sensitivity, 97% specificity, low positive predictive value, and high negative predictive value for microscopy. An ELISA kit (TechLab®) that was specific for E. histolytica detected a 3.1% (43/1403) prevalence for E. histolytica infection. Conclusions: The ELISA kit (IVD®) can be used as an alternative screening tool. The high prevalence of E. histolytica infection detected in this study warrants the implementation of actions directed toward health promotion and preventive measures. .

Detección de Entamoeba histolytica y Entamoeba dispar mediante PCR, en niños menores de cinco años con diarrea, en Maracaibo, Venezuela: Estudio preliminar / Detection of Entamoeba histolytica and Entamoeba dispar by PCR in children, less than five years of age with diarrhea, in Maracaibo, Venezuela: A preliminary study

Para determinar la prevalencia de Entamoeba histolytica como productora de diarrea, se realizó un estudio en niños menores de cinco años con diarrea que asistieron a diversas consultas del Servicio Autónomo Hospital Universitario de Maracaibo, Venezuela. A las muestras de heces obtenidas, se les realizó examen macroscópico, microscópico con solución salina fisiológica 0,85% y lugol, así como coloración de Kinyoun. El resto de la muestra se congeló hasta la extracción del ADN y luego se amplificaron mediante PCR separadas para E. histolytica y E. dispar. De las 50 muestras analizadas mediante examen microscópico, ninguna presentó trofozoítos y/o quistes de Entamoeba histolytica/dispar/moshkovskii, ni coccidios intestinales. Los parásitos detectados fueron Giardia lamblia (6%), Blastocystis sp. (6%), Pentatrichomonas hominis (2%), Ascaris lumbricoides (2%) y Trichuris trichiura (2%). Mediante PCR, 6 muestras (12%) presentaron ADN de E. dispar y 2 (4%) ADN de E. histolytica. Ningún niño presentó asociación de ambas amibas. Los dos niños que presentaron E. histolytica tenían 1 año de edad. E. dispar si fue detectada en niños de menor edad. Se sugiere que la prevalencia de E. histolytica en niños menores de cinco años con diarrea es realmente baja.

To determine the prevalence of Entamoeba histolytica as a producer of diarrhea, a study was conducted in children, less than five years of age, with diarrhea who attended several out patient clinics of the Servicio Autónomo Hospital Universitario, Maracaibo, Venezuela. A macroscopic and microscopic examination with physiological saline, lugol and Kinyoun staining were performed to the stool samples obtained. The remainder of the sample was frozen until DNA extraction, and PCR amplification was performed separately for E. histolytica and E. dispar. Microscopic examination showed no trophozoites and/or cysts of Entamoeba histolytica/dispar/moshkovskii, or intestinal coccidians in any of the 50 samples analyzed. Parasites detected were Giardia lamblia (6%), Blastocystis sp. (4%), Pentatrichomonas hominis (2%), Ascaris lumbricoides (2%) and Trichuris trichiura (2%). By PCR, six samples (12%) had DNA of E. dispar and two (4%) had DNA from E. histolytica; no child showed association of both amoebae. The two children who had E. histolytica were one-year-old. E. dispar was detected in younger children. We suggest that the prevalence of E. histolytica in children under five years is really low.

High occurrence of Entamoeba histolytica in the municipalities of Ariquemes and Monte Negro, State of Rondônia, Western Amazonia, Brazil / Alta ocorrência de Entamoeba histolytica nos municípios de Ariquemes e Monte Negro, estado de Rondônia, Amazônia Ocidental, Brasil

Introduction: Entamoeba histolytica infections were investigated in residents of the Ariquemes and Monte Negro municipalities in Rondônia State, Brazil. Methods: Stool samples of 216 individuals were processed by the spontaneous sedimentation method and analyzed by microscopy for detection of the E. histolytica/E. dispar complex, followed by the immunoassay method using an enzyme-linked immunosorbent assay-based kit for the E. histolytica stool antigen. Results: E. histolytica/E. dispar cysts were present in 61% (50/82) and 44% (59/134) of the samples from Ariquemes and Monte Negro respectively, with a significant difference in the occurrence of infection between the two populations [p < 0.05; χ2 = 5.2; odds ratio = 2.0 (1.1 - 3.6)]. The E. histolytica antigen detection rate was 36.6% (30/82) for stool samples from Ariquemes, and 19.4% (26/134) for stool taken from the residents of Monte Negro. The rate of the occurrence of amoebiasis was significantly higher in the population from Ariquemes [p < 0.05; χ2 = 7.8; odds ratio = 2.4 (1.2 - 4.7)]. Discussion: Due to the high occurrence of E. histolytica infected residents diagnosed in the region and the unavailability in local clinics of a test to distinguish between the two Entamoeba species, physicians should consider treating E. histolytica/E.dispar infections. Conclusion: The results indicate that E. histolytica infection is highly endemic in the studied areas. .

Introdução: Infecções por Entamoeba histolytica foram investigadas em moradores dos municípios de Ariquemes e Monte Negro, Rondônia, Brasil. Métodos: Amostras de fezes de 216 indivíduos foram processadas por microscopia óptica para detecção de cistos do complexo E. histolytica/E. dispar, seguido pelo método de imunoensaio utilizando kit de ensaio imunoenzimático para detecção específica de antígeno de E. histolytica. Resultados: Cistos de E. histolytica/E. dispar estavam presentes em 61% e 44% das amostras de Ariquemes e Monte Negro, respectivamente com diferença significativa na ocorrência da infecção entre as duas populações [p < 0,05; χ2 = 5,2; Odds relativa = 2,0 (1,1 - 3,6)]. A taxa de detecção de antígenos de E. histolytica nas amostras provenientes de Ariquemes foi de 36,6% e de 19,41% nas amostras de Monte Negro, sendo a ocorrência de amebíase significativamente maior na população de Ariquemes [p < 0,05; χ2 = 7,8; Odds relativa = 2,4 (1,2 - 4,7)]. Discussão: A elevada frequência da infecção por E. histolytica em residentes na região, bem como a indisponibilidade de avaliação clínica por testes específicos para distinção entre as duas espécies de Entamoeba, deve promover uma reflexão sobre o tratamento de infecções pelo complexo E. histolytica/E. dispar. Conclusão: Nas populações avaliadas foram detectadas elevadas ocorrências de E. histolytica. .

Prevalence and characters of Entamoeba histolytica infection in Saudi infants and children admitted with diarrhea at 2 main hospitals at south Jeddah: a re-emerging serious infection with unusual presentation

In this study, Entamoeba histolytica had high prevalence and unusual presentation by affecting high proportion of infants under 1 year; severe clinical manifestations, and laboratory findings that were known to be usually encountered in invasive amebiasis as significant leukocytosis for age, neutrophilic leukocytosis for age, and positive C-reactive protein were found among more than 50% of admitted Saudi infants and children with E. histolytica infection in our locality. E. histolytica can be a re-emerging serious infection when it finds favorable environmental conditions and host factors which are mainly attributed to inadequate breastfeeding in this study. This may occur in any other area of the world with the same risk factors, so we must be ready to tackle it with effective and more powerful preventive measures.

Breast-Feeding Protects Infantile Diarrhea Caused by Intestinal Protozoan Infections

This study investigated the effect of breast-feeding in protection against protozoan infection in infants with persistent diarrhea. Infants were classified into 2 groups; 161 breast-fed infants and the same number of non-breast-fed infants. Microscopic examinations of stool were done for detection of parasites and measuring the intensity of infection. Moreover, serum levels of IgE and TNF-alpha were measured by ELISA. Cryptosporidium spp., Entamoeba histolytica/Entamoeba dispar, Giardia lamblia, and Blastocystis sp. were demonstrated in infants with persistent diarrhea. The percentage of protozoan infections was significantly lower in breast-fed infants than that in the non-breast-fed infants. The levels of IgE and TNF-alpha were significantly lower in the breast-fed group than in the non-breast-fed group. There were significant positive associations between the serum levels of IgE and TNF-alpha and the intensity of parasite infection in the breast-fed group. It is suggested that breast-feeding has an attenuating effect on the rate and intensity of parasite infection.

Nested PCR reveals elevated over-diagnosis of E. histolytica in Barcelona, Venezuela / Nested PCR revela elevado sobrediagnóstico de E. histolytica en Barcelona, Venezuela

The aim of this study was to identify the presence of Entamoeba histolytica and E. dispar by nested PCR in children attending the “Dr. Luis Razetti” Hospital, Barcelona, Anzoátegui State. Of the 1,141 fecal samples coproparasitologically evaluated by conventional microscopy, 150 were diagnosed positive for E. histolytica in 0-10 year-old-children, of both sexes. The signs, symptoms and a full coproparasitological report were obtained from all of these and nested PCR was performed to identify E. histolytica and E. dispar. The conventional microscopy results showed a diagnostic frequency of E. histolytica in 13.2% of the cases, of which 79.3% were positive only for this pathogen. However, nested PCR showed that of these, only 28% (42/150) were actually infected by Entamoeba spp., revealing a high over-diagnosis of E. histolytica. We also identified 9.3% E. histolytica, 4% E. dispar and 4.7% mixed infections. Diarrhea was the most common symptom, followed by abdominal pain and fever. Bloody stools were statistically associated with E. histolytica, but were also found for E. dispar infections. This study demonstrates that molecular techniques complementary to conventional methods enable the correct identification of Entamoeba spp., thus contributing to an improved epidemiological assessment of these parasites and implementation of the appropriate treatment.

Esta investigación planteó detectar por nested PCR Entamoeba histolytica y E. dispar en niños del Hospital “Dr. Luis Razetti” de Barcelona, estado Anzoátegui y su asociación con síntomas clínicos. De 1.141 muestras fecales evaluadas parasitológicamente por microscopía convencional, 150 fueron positivas a E. histolytica en niños de 0-10 años y de ambos sexos. Se obtuvo información de signos, síntomas y reporte parasitólogico completo de cada uno de los pacientes y se realizó nested PCR para identificar E. histolytica y E. dispar. Los resultados de la microscopía convencional demostraron una frecuencia de diagnóstico de E. histolytica del 13,2%. En el 79,3% de estas positivas se reportó esta especie como único patógeno. Sin embargo, la nested PCR evidenció que sólo 28,0% (42/150) de las mismas presentaron infecciones por Entamoeba, evidenciándose un elevado sobrediagnóstico de E. histolytica. Además se identificaron 9,3% infecciones por E. histolytica, 4,0% E. dispar, y 4,7%infecciones mixtas. La diarrea fue el síntoma más común, seguido de dolor abdominal y fiebre. La presencia de sangre demostró asociación estadísticamente significativa con E. histolytica, pero también se reportó en infecciones por E. dispar. Este estudio demuestra que las técnicas moleculares complementarias a los métodos convencionales, permiten la identificación correcta de especies de Entamoeba, contribuyendo con una mejor evaluación epidemiológica de estos parásitos y la aplicación adecuada del tratamiento.

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.

Evaluation of small-subunit rRNA touchdown polymerase chain reaction for direct detection of Entamoeba histolytica in human pus samples from patients with amoebic liver abscess.

Purpose: The aim of the present study was to evaluate the use of touchdown polymerase chain reaction (TD-PCR) for the detection of Entamoeba histolytica in liver pus samples obtained from patients with a clinical diagnosis of amoebic liver abscess (ALA) using small-subunit rRNA (SSU rRNA) as the target gene. Materials and Methods: Microscopic examination in vitro culture and serological test for the detection of E. histolytica in 67 pus samples obtained from ALA patients was performed. Molecular studies were carried out by both conventional PCR and TD-PCR targeting the SSU rRNA gene using the same sets of primers and the results were compared. Results: TD-PCR detected the presence of E. histolytica in 86.5% of the liver pus samples within 2.5 h as compared with 82.08% by conventional PCR within 3.5-4 h. Conclusion: TD-PCR assay may serve as a relatively better detection method for E. histolytica over conventional PCR with respect to the turnaround time, increased sensitivity, specificity and yield.

Hepato-pulmonary amebiasis: a case report

Infections with Entamoeba histolytica are seen worldwide and are more prevalent in the tropics. About 90 percent of infections are asymptomatic, and the remaining 10 percent produce a spectrum of clinical syndromes, ranging from dysentery to abscesses of the liver or other organs. Extra-intestinal infection by E. histolytica most often involves liver. Pleuro-pulmonary involvement, seen as the second most common extra-intestinal pattern of infection, is frequently associated with amebic liver abscess. Pulmonary amebiasis occurs in about 2-3 percent of patients with invasive amebiasis. We report herein the case of a 45-year-old male presenting with hepato-pulmonary amebiasis. The diagnosis was established from direct examination of sputum, in which trophozoites of E. histolytica were detected, and by serology. Following treatment with metronidazole and chloroquine, the clinical evolution improved significantly. On regular follow-up visits, the patient was asymptomatic. This case report reiterates the need for collaboration between clinicians and microbiologists for timely diagnosis of such infections.

Neonatal amoebiasis.

The authors report a case of neonatal amebiasis presenting with vomiting, refusal to feeds, abdominal distension and mucoid stools. The diagnosis was made on the basis of clinical appearance of stools and the presence of Entameba Histolytica. The purpose of this report is to alert to the possibility of amoebiasis in newborns, if baby presents with NEC like picture in an endemic area and to send the stool sample immediately to examine for trophozoites, especially because the diarrheal stool starts autolyzing within 30 min of defecation. This 14-day-old newborn, who was possibly infected with orally given jaggery solution is presented. He was successfully treated with intravenous tinidazole.

Absceso hepático amebiano en el embarazo: reporte de un caso / Amebic liver abscess in pregnancy: a case report

La asociación de absceso hepático amebiano y embarazo es poco frecuente y se asocia a un aumento de la morbilidad y mortalidad materna. Presentamos el caso de una paciente de 33 años, cursando un embarazo de 35 semanas, que ingresó a nuestro servicio con el diagnóstico de absceso hepático, cuya etiología fue E. histolytica.

The appearance of amebic liver abscess in pregnancy is uncommon and is associated to increased maternal morbidity and mortality. We report the case of 33 years old woman, with a 35 weeks pregnancy that was admitted in our Hospital with the diagnosis of liver abscess, whose etiology was E. histolytica.

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.

[Differentiation of Entamoeba histolytica and Entamoeba dispar by single polymerase chain reaction]

In most part of the world detection of cysts and trophozoites of Entamoeba is based on morphological structure of this species in stool sample by microscopy. However, microscopic examination is unable to distinguish between similar morphological protozoa such as Entarnoeba histolytica and Entamoeba dispar. A simple and cost-effective method is needed in medical laboratories for detection and differentiation of these two species. Stool samples of patients who were referred from health care centers were examined by direct microscopy and trichrome stain. Polymerase chain reaction [PCR] utilizing pEd30F and pEd21AS primers from Peroxiredoxin gene, was used for differentiation of E. histolytica and E. dispar. Genomic DNA from samples was amplified by these primers. The fragment under 100 bp was related to E. histolytica and in contrast the fragment above the 100 bp was related to E. dispar. In this study from 22 microscopic positive samples, E. histolytica was observed only in one patient and E. dispar was detected in the other 21 samples. The result of this study indicate that the PCR reaction could amplify E. dispar and E. histolytica with just one primer pair and this is a cost-effective method for distinguishing between these two species

Detection of Entamoeba histolytica DNA in the saliva of amoebic liver abscess patients who received prior treatment with metronidazole.

Saliva is an easily-accessible and a non-invasive clinical specimen alternate to blood and liver pus. An attempt was made to detect Entamoeba histolytica DNA released in the saliva of amoebic liver abscess (ALA) patients by applying 16S-like rRNA gene-based nested multiplex polymerase chain reaction (NM-PCR). The NM-PCR detected E. histolytica DNA in the saliva of eight (28.6%) of 28 ALA patients. The NM-PCR result was negative for E. histolytica DNA in the saliva of all the eight ALA patients who were tested prior to treatment with metronidazole but was positive in the saliva of eight (40%) of 20 ALA patient who were tested after therapy with metronidazole. The NM-PCR detected E. histolytica DNA in liver abscess pus of all 28 (100%) patients with ALA. The TechLab E. histolytica II enzyme-linked immunosorbent assay was positive for E. histolytica Gal/GalNAc lectin antigen in the liver abscess pus of 13 (46.4%) of the 28 ALA patients. The indirect haemagglutination (IHA) test was positive for anti-amoebic antibodies in the serum of 22 (78.6%) of the 28 ALA patients and 2 (5.7%) of 35 healthy controls. The present study, for the first time, demonstrates the release of E. histolytica DNA in the saliva of ALA patients by applying NM-PCR.

Amebic cerebral abscess mimicking bacterial meningitis.

We report a case of an amebic brain abscess in a 2-year-old girl, with symptoms mimicking bacterial meningitis with no evidence of disease elsewhere. Histological evaluation of the abscess revealed the organisms, and the abscess regressed in response to specific medical treatment. This article reviews the rarity of these abscesses and difficulty in the diagnosis.

Enteric protozoan parasitic infection in HIV seropositive individuals with diarrhoea.

A total of 78 HIV seropositive patients were included in the study from January 2004 to March 2006. Enteric protozoan parasitic infections were diagnosed by microscopic examination of stool by fresh wet mount, stool smears stained by modified Z.N., Trichrome and Calcofluor white stains. 20 samples (25.6%) revealed presence ofenteric protozoan parasites.