Nested PCR reveals elevated over-diagnosis  of E. histolytica in Barcelona, Venezuela. 

Hectorina Rodulfo ¹, Beatriz Ahmar ¹, María E. Rodríguez ¹, Leonor Mora ² y Marcos De Donato ¹. 

¹ Laboratorio de Genética Molecular, Instituto de Investigaciones en Biomedicina y Ciencias Aplicadas “Dra. Susan Tai”

² Departamento de Bioanálisis. Universidad de Oriente, Núcleo de Sucre. Cumaná, Venezuela.

Corresponding author: Marcos De Donato. Lab. Genética Molecular, IIBCA, Universidad de Oriente. Av. Universidad, Cerro del Medio, Cumaná, Venezuela. Tel: +58-293-4175285, Fax: +58-293-4521295. E-mail: marcosdedonato@yahoo.com 

Abstract.

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.

Nested PCR revela elevado sobrediagnóstico de E. histolytica en Barcelona, Venezuela.

Resumen.

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.

Recibido: 13-06-2012. Aceptado: 22-11-2012

INTRODUCTION 

Entamoeba histolytica is the causal agent of intestinal amoebiasis, one of the principal causes of mortality in humans worldwide (1-4). Other Entamoeba species, such as E. dispar and E. moshkovskii have also been found in patients with gastrointestinal symptoms (5-9). However, there is as yet no definitive evidence demonstrating that these two species are pathogenic to humans (10-12). 

Clinical features of amebiasis range from asymptomatic colonization to amoebic dysentery and invasive extra intestinal amoebiasis, the latter in some cases in the form of liver abscesses (13). An estimated 50 million people suffer from this invasive disease worldwide, producing an annual death toll of between 40,000 and 100,000 (14, 15). Although this parasite is distributed throughout the world, prevalences exceeding 10% have only been reported from some developing countries (16). Despite the availability of effective treatments against E. histolytica, morbidity and mortality rates have persisted, suggesting that measures to eliminate or limit the disease are at present largely ineffective. Nevertheless, as humans are apparently the only hosts, an appropriate control program should be able to eradicate the infection (17, 18). 

E. histolytica infections are diagnosed based on the study of the clinical symptoms, as well as the microscopic examination of serial stool samples. The latter is a faster and easier procedure, but its sensitivity is limited and requires an experienced observer to accurately distinguish between pathogenic and non-pathogenic species; a serious cause of error for the correct diagnosis of the disease. For this reason, direct diagnosis by itself is considered to be insufficient for the identification of E. histolytic and should be complemented by fixation techniques such as permanent trichrome staining that permit the intracellular elements to be visualized more easily (19,20). Haque et al. (1), however, argue that trichrome and iron hematoxylin staining are not good methods for the detection of E. histolytica, because they do not differentiate between this and other non-pathogenic species which are morphologically identical. 

The diagnosis of false negatives of E. histolytica is thus at least partly due to a delay in sample processing, short analysis time, analyses performed by technicians without adequate theoretical and practical training and a lack of complementary methods that help to improve the visualization of hematophagous trophozoites (21). False positives, on the other hand, occur due to the incorrect identification of non-pathogenic species of amoeba and host cells such as macrophages (22-24). The difficulties of differentiating E. histolytica from other Entamoeba spp. is, in many cases, the reason why the prevalence of this infection vary so greatly from one region to another (5). 

In order to provide a solution to the problems of the diagnosis of Entamoeba related infections, rapid and highly sensitive Polymerase Chain Reaction (PCR)-based protocols have been developed, and although several studies have compared the different methods of species-specific diagnoses (25-27), molecular techniques have been effective for the successful detection and differentiation of E. histolytica and E. dispar in clinical samples. Parija and Khaimar (7) analyzed 746 stool samples with cysts and trophozoites of E. histolytica, E. dispar and E. moshkovskii, using PCR to amplify the small rRNA subunit. These authors found a greater prevalence of E. dispar (8.8%) compared to E. histolytica (1.7%). E. histolytica was, in fact, only actually present (as diagnosed by PCR) in 19% of the 68 stool samples registered as containing E. histolytica by microscopic examination, implying that 81% of suspected infections were wrongly diagnosed and patients were thus unnecessarily treated with antiamoebic medication. This over-diagnosis of E. histolytica, has led to a re-evaluation of the epidemiology of amoebiasis in terms of prevalence and morbidity, particularly in geographical areas with high endemicity (19,28). 

In Venezuela, 6,872,282 cases of diarrhea, mainly in children, were reported between 2007 and 2010. Of these, 574,225 were the result of amoebiasis, with Zulia State having the largest number of cases and Anzoátegui and Sucre being the most affected states in the eastern region of the country (29). In addition, changes in the frequencies of the E. histolytica/E.dispar complex have been reported, with high prevalences in different regions (30-33). The application of PCR by Rivero et al. (34) in Maracaibo (Western Venezuela), and Mora et al. (35), in Cumaná (Eastern Venezuela), have shown significant differences in the frequencies of infections by species of Entamoeba between the microscopic and molecular detection. 

The fact that an important number of infections by Entamoeba spp. has been reported in Venezuela, means that more appropriate, sensitive and specific methods that allow for the correct detection and differentiation of species within this genus should be implemented. We thus undertook this investigation in order to assess the real frequencies of E. histolytica and E. dispar in stool samples diagnosed as positive for E. histolytica and/or E. dispar by conventional methods in patients from Barcelona, Venezuela. 

METHODOLOGY 

Study population 

A total of 150 stool samples collected from children aged 0-10 of both sexes, between January and August 2009, who attended the Pediatric Department “Dr. Rafael Tobías Guevara” at the Dr. “Luis Razetti” Hospital, Barcelona, Anzoátegui State, were diagnosed as microscopically positive for Entamoeba histolytica, when they were observed using wet preparations with 0.85% physiological saline solution and Lugol’s solution. The physical characteristics of the samples such as: appearance, consistency, color, odor, pH, presence or absence of mucus, blood and/or adult worms, were noted as well as the signs and symptoms of the children at the time of the collection of the sample (fever, diarrhea, abdominal pain, among others). In addition, intestinal worms and protozoa were detected and identified microscopically in each of the samples. 

The consent of the legal representatives of the children who participated in the study was sought in each case after informing them about the aims of the research. All of the methods used were previously approved by the Bioethics and Biosafety Committee at the Instituto de Investigaciones en Biomedicina y Ciencias Aplicadas, Universidad de Oriente (IIBCAUDO), Cumaná, Venezuela. Once the representative had authorized the participation of their child or children and the stools had been processed by microscopy, l g of fecal matter was removed from each sample and placed in a 1.5 mL sterile Eppendorf tube with 500 µL phosphate buffered saline (PBS) 1X. The tubes were then transported on ice to the Molecular Genetics Laboratory, IIBCA-UDO, where they were stored at –20°C until analysis by PCR.

DNA extraction 

Genomic DNA was extracted from the 150 stool samples using the Promega Wizard® Genomic DNA purification Kit, employing the protocol suggested by the manufacturer with the following modifications: 200 µL of each of the fecal samples preserved in PBS were placed in a 1.5 mL Eppendorf tube, centrifuged at 14 000 g for 3 min at 18°C and the supernatant discarded. Then 400 µL nuclei lysis solution was added and the mixture homogenized by pipetting the sediment. Cyst walls were fractured by sonication, using a high intensity sonicator (Autotone Ultrasonic) set at the minimum speed for 2 sec and repeated three times, with the samples held on ice. 10 µL proteinase K was then added. Samples were incubated for 2 hours at 65°C and then left to cool for 5 min at room temperature. The extraction procedure was then continued using the protocol recommended by the kit and the DNA obtained stored at –20°C, until its subsequent amplification by PCR. 

(PCR) polymerase chain reaction 

The Nested PCR procedure has been proposed by the International Centre for Diarrhoeal Disease Research, Dhaka, Bangladesh (ICDDR, B) (36), as well as the Jawaharlal Institute of Postgraduate Medical Education and Research (JIPMER), Puducherry, India (7) as the optimal method for the detection and differentiation of E. histolytica and E. dispar, using the 16S rRNA-like gene sequence as the genomic target. 

Nested PCR was performed by first amplifying a fragment specific to the Entamoeba genus (E1-E2 primers 898 bp) and afterwards the fragments specific to E. histolytica and E. dispar (439 bp, and174 bp, respectively) following the protocol described by Parija and Khairnar (7). A cultivated strain of E. histolytica; IULA-0593:2 (NER), donated by the Immunology Institute, Universidad de los Andes (ULA), Mérida, Venezuela; and a positive sample for E. dispar, UDO402 strain (35), were used as positive controls in each PCR run. The amplified products were separated by electrophoresis on a 2% agarose gel (EC330 Primo Minicell Gel Electrophoresis System) in Tris-boric acid-EDTA (TBE) buffer 1X at 80 V for 45 min, stained with ethidium bromide (0.5 µg/mL) and visualized in a ultraviolet transilluminator. The molecular marker used was the Axygen100 pb ladder. 

Statistical analysis 

The results obtained are shown in the tables and figures. Associations between the Entamoeba species assessed in this study with the age, sex and symptoms of the patients and the macroscopic characteristics of the fecal material, were tested with the Chi-square (c²) test (37, 38), using version 11.5 of the SPSS statistical package. 

RESULTS 

E. histolytic was found in 13.2% (150) of the 1,141 stool samples analyzed microscopically by bioanalysts in the Pediatric Department Laboratory at the “Dr. Luis Razetti" Hospital, Barcelona, Anzoátegui State, of which 79.3% contained cystic forms and trofozoites of E. histolytica (Table I). In addition, in 19.3% of the samples, contained forms of other intestinal protozoa such as Giardia duodenalis, Blastocystis hominis, E. coli and Chilomastix mesnili; and in 1.4%, co-infections with the eggs of intestinal helminthes, such as Trichuris trichiura and Hymenolepis nana, were found (Table I), demonstrating a high prevalence of infections caused by intestinal protozoa in the samples analyzed. However, it must be emphasized that these samples were not concentrated as part of the clinical diagnosis, thus the frequency of helminths and other intestinal protozoa observed may have been underestimated. 

TABLE I. FREQUENCY OF CHILDREN INFECTED WITH E. histolytica ONLY AND E. histolytica ASSOCIATED WITH OTHER INTESTINAL PROTOZOA AND HELMINTHES FOR EACH SYMPTOM OBSERVED 

A total of 98.7% of the children showed symptoms related to the infection, with acute diarrhea being the most prevalent symptom (68%), followed by fever (16%) and abdominal pain (12.7%) (Table I). When conventional microscopy was used as the diagnostic method, E. histolytica was identified as single infection in 79.3% of symptomatic and asymptomatic children and in only 12% of samples they were accompanied by other protozoa and pathogenic intestinal helminths. In 8.7% of samples, E. histolytica was found causing infections together with commensal protozoa such as E. coli and C. mesnili (Table I). 

The first round of amplification of the nested PCR, corresponding to the 898 bp fragment specific for Entamoeba spp., allowed us to detect a 28% (42/150) infection rate by any of the Entamoeba species. The second round of amplification produced species specific fragments in all of these samples, of which 29 (19.3%) were identified as E. histolytica, 6 (4%) as E. dispar, and 7 (4.7%) as mixed infections by E. histolytica/E.dispar (Eh/Ed) (Figs. 1 and 2).

Fig. 1. Representative gels for the second round of the nested PCR showing the presence of E. histolytica as diagnosed from stool samples collected from the studied children. M: 100 bp molecular weight marker (Axygen). C+: E. histolytica NER strain positive control: 439 bp. Evaluated stool samples: wells 1-74. C-: negative control.

Fig. 2. Representative gels for the second round of the nested PCR showing the presence of E. dispar as diagnosed from stool samples collected from the studied children. M: 100 bp molecular weight marker (Axygen). C+: E. dispar UDO402 strain positive control: 174bp. Evaluated stool samples: wells 1-74. C-: negative control.

The PCR procedure applied to the 150 stool samples microscopically diagnosed as positive for E. histolytica showed that microscopic analysis over-diagnosed the incidence of amebiasis by 72% (108/150). As already mentioned above, PCR showed that only 42 of the 150 samples were, in fact, positive for the Entamoeba species diagnosed in this investigation. 

Furthermore, PCR revealed that in 89.7% (26/29) of infections, E. histolytica was the only parasite involved, whereas in 10.3% (3) of the cases there was a co-infection with E. coli and C. mesnili. In those infections caused by E. dispar and mixed Eh/Ed infections no other parasites were found, except for a mixed infection with E. coli and C. mesnili. It should be noted that E. histolytica was not detected in samples where it had been microscopically diagnosed as co-infecting with other protozoa and pathogenic intestinal helminthes. This could be due to the difficulty of differentiating Entamoeba spp. from host cells when using SSF and Lugol´s solution as the staining agents during parasitological diagnosis. 

Regarding the symptoms associated with the infections (Table II), diarrhea was also the most common symptom of infection by E. histolytica (62.1%), followed by abdominal pain (20.7%) and fever (13.8%). Diarrhea was also present in 66.6% of all E. dispar and mixed Eh/Ed infections, and 33.3%of children infected by E. dispar also suffered fever. These observations thus show that both E. histolytica and E. dispar may be found in the same group of individuals with intestinal symptoms.

TABLE II. FREQUENCY OF THE SYMPTOMS SHOWN BY THE CHILDREN UNDER STUDY FOR EACH OF THE INFECTING AGENTS AS DIAGNOSED BY PCR 

Eh: Entamoeba histolytica.    Ed: Entamoeba dispar. M: mixed Eh/Ed infection. 

We could not find any species specific pattern as regards stool consistency when we macroscopically examined the PCR positive stool samples (Table III), as both soft and liquid stools were infected by either species. Mixed infections did, however, predominate in liquid samples (71.4%). Similarly, the presence of blood was not necessarily indicative of pathogenic species, although it was statistically associated with E. histolytica (c² = 4.753; P < 0.05) (Table IV). Lastly, mucus was found in the fecal samples of most of the E. histolytica, E. dispar and mixed Eh/Ed infections (Table III). 

TABLE III. MACROSCOPIC CHARACTERISTICS OF THE PCR-POSITIVE AND NEGATIVE STOOL SAMPLES COLLECTED FROM THE CHILDREN UNDER STUDY 

DISCUSSION 

In general, infections caused by intestinal protozoa predominated in the children examined. In this regard, authors such as Ouattara et al. (39) indicate that polyparasitism by intestinal protozoa is frequent (80.2%) in the 6-16 year-old age-group, with the most common species being, Endolimax nana, E. coli, G. duodenalis and the Eh/Ed complex; this last with a prevalence of 11.3%. This confirms the presence and range of these micro-organisms and indicates that an understanding of their distribution in areas of transmission of pathogenic protozoa could aid the development of disease control programs that combine treatment with prevention. 

With respect to the prevalence of E. histolytica in Venezuela, large variations in different regions of the country have been reported. In the West, a prevalence of 4% for the E. histolytica/E. dispa rcomplex was detected by parasitological analysis (33). A study of the Yukpa ethnicity in rural areas of Zulia state, showed a prevalence of 21.9% E. histolytica infections in children aged 0-14 (32). In addition, Rivero et al. (34), obtained a prevalence of 10.8% E. histolytica infections in Maracaibo, Zulia state using PCR, compared with that of 20.6% mixed E. histolytica/E. dispa rinfections diagnosed by parasitological analysis. These researchers found no significant associations between infection by these species and age and/or sex. 

In Bolívar state (southeast Venezuela), Devera (40) reported the absence of this intestinal protozoa after microscopic analysis. However, Mora et al. (35) found a prevalence of 5.4% for E. histolytica and 3.5% for E. dispar infections using molecular detection by PCR in individuals with gastrointestinal symptoms in Cumaná, Sucre state. According to recent reports there is a higher prevalence of E. histolytica in Sucre state, compared to other states. This coincides with our results for Barcelona, Anzoátegui state, showing higher frequencies in Northeastern Venezuela, compared to the Southeastern area. 

Our study agrees with that of Mora et al. (35), in that both indicate an important over-diagnosis of pathogenic species by parasitological methods. They reported 51.2% false positives from conventional microscopic analysis. On the contrary, Rivero et al. (34) found that under-diagnosed by 10.6% by microscopic examination: 42 positive samples were detected by this method compared to 47 positives by PCR, however only 22 (10.78%) of these were identified as E. histolytica. 

The tendency of microscopic analysis to produce false results due to confusion between macrophages and trofozoites, and polymorphonuclears (PMN) and cysts, leading to the incorrect identification of amoebas, is well known (19, 22, 23). Thus, the current recommendation when species specific diagnosis is not possible is to report: “E. histolytica/E. dispar/E. moshkovskii” in order to describe the presence of species with identical morphologies in stool samples. Researchers and technicians are also encouraged to use new technologies wherever possible in order to elucidate the true epidemiology and pathogenesis of Entamoeba spp., including the least studied E. moshkovskii (41). 

The fact that 108 samples were diagnosed by conventional microscopy as positive for E. histolytica, but were found to be negative for this species by PCR, may be explained by the presence of intestinal amoebas morphologically similar to E. histolytica such as E. hartmanni and E. polecki. This last, although it is a parasite of monkeys and pigs, and is uncommon in humans, has been reported from the latter on eight occasions in Venezuela (42-45). E. hartmanni, on the other hand, has often been reported from western regions of Venezuela since 1976, using staining and concentration techniques (31, 45, 46-50). All these studies have pointed out that E hartmanni may be less frequently detected in fresh stools or concentrated samples due to its small size, or because its morphological features make it indistinguishable from other Entamoeba species, thus hindering a specific diagnosis. 

The macroscopic characteristics of the stool are important to indicate the correct diagnostic methodology to use in the search for intestinal pathogens. In this study, however, the characteristics of PCR-positive samples (consistency and presence of mucus) were not reliable indicators of the presence of the different Entamoeba spp. Nevertheless, the presence of blood, although not exclusive to one pathogenic species, did show a statistically significant association with E. histolytica. Different research papers have pointed out that, while E. dispar is considered to be a commensal amoeba, it has also been identified in patients with gastrointestinal symptoms (5, 9, 38, 51). We now know that hematophagism is not exclusive to pathogenic amoebae, since it has been demonstrated that E. dispar cells from stools samples can contain red blood cells inside, which has been corroborated by in vitro studies, showing the ability of E. dispar to ingest red blood cells (5, 53). 

The difficulties of correctly identifying Entamoeba spp. has led to the use of PCR-based procedures as the method of choice for clinical and epidemiological studies in developed countries (54-57), a practice strongly endorsed by the World Health Organization. The use of molecular biology techniques has enabled the identification of E. histolytica in a variety of clinical specimens, including feces, tissue, liver abscesses and aspirates (19). 

Lastly, according to our experience at the Molecular Genetics Laboratory (LGM) at the IIBCAUDO, the application of PCR for the diagnosis of intestinal amoebiasis, as well as more sensitive and rapid molecular techniques (58,59) that are less expensive and can be applied in the field (60), provides a complementary method to the conventional protocols that enables the correct identification of Entamoeba spp. Thus, PCR provides us with an extremely useful tool for a better understanding of the biology, diagnosis and epidemiology of these species in different regions. 

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