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Redalyc.Nested PCR Reveals Elevated Over-Diagnosis of E. Histolytica In Investigación Clínica ISSN: 0535-5133 [email protected] Universidad del Zulia Venezuela Rodulfo, Hectorina; Ahmar, Beatriz; Rodríguez, María E.; Mora, Leonor; De Donato, Marcos Nested PCR reveals elevated over-diagnosis of E. histolytica in Barcelona, Venezuela. Investigación Clínica, vol. 53, núm. 4, octubre-diciembre, 2012, pp. 365-377 Universidad del Zulia Maracaibo, Venezuela Available in: http://www.redalyc.org/articulo.oa?id=372937690006 How to cite Complete issue Scientific Information System More information about this article Network of Scientific Journals from Latin America, the Caribbean, Spain and Portugal Journal's homepage in redalyc.org Non-profit academic project, developed under the open access initiative Invest Clin 53(4): 365 - 377, 2012 Nested PCR reveals elevated over-diagnosis of E. histolytica in Barcelona, Venezuela. Hectorina Rodulfo1, Beatriz Ahmar1, María E. Rodríguez1, Leonor Mora2 y Marcos De Donato1. 1Laboratorio de Genética Molecular, Instituto de Investigaciones en Biomedicina y Ciencias Aplicadas “Dra. Susan Tai”, 2Departamento de Bioanálisis. Universidad de Oriente, Núcleo de Sucre. Cumaná, Venezuela. Keywords: Entamoeba, diagnosis, PCR, E. histolytica. 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 ob- tained from all of these and nested PCR was performed to identify E. histolytica and E. dispar. The conventional microscopy results showed a diag- nostic 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 associ- ated with E. histolytica, but were also found for E. dispar infections. This study demonstrates that molecular techniques complementary to conven- tional methods enable the correct identification of Entamoeba spp., thus con- tributing to an improved epidemiological assessment of these parasites and implementation of the appropriate treatment. Corresponding author: Marcos De Donato. Lab. Genética Molecular, IIBCA, Universidad de Oriente. Av. Univer- sidad, Cerro del Medio, Cumaná, Venezuela. Tel: +58-293-4175285, Fax: +58-293-4521295. E-mail: [email protected] 366 Rodulfo y col. Nested PCR revela elevado sobrediagnóstico de E. histolytica en Barcelona, Venezuela. Invest Clin 2012; 53 (4): 365 - 377 Palabras clave: Entamoeba, diagnóstico, PCR, E. histolytica. Resumen. Esta investigación planteó detectar por nested PCR Entamoe- ba histolytica y E. dispar en niños del Hospital “Dr. Luis Razetti” de Barcelo- na, estado Anzoátegui y su asociación con síntomas clínicos. De 1.141 mues- tras 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 comple- to de cada uno de los pacientes y se realizó nested PCR para identificar E. histolytica y E. dispar. Los resultados de la microscopía convencional demos- traron 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 embar- go, la nested PCR evidenció que sólo 28,0% (42/150) de las mismas presen- taron infecciones por Entamoeba, evidenciándose un elevado sobrediagnósti- co de E. histolytica. Además se identificaron 9,3% infecciones por E. histolyti- ca,4,0%E. dispar, y 4,7%infecciones mixtas. Ladiarreafueelsíntomamás común, seguido de dolor abdominal y fiebre. La presencia de sangre demos- tró asociación estadísticamente significativa con E. histolytica, pero también se reportó en infecciones por E. dispar. Este estudio demuestra que las téc- nicas moleculares complementarias a los métodos convencionales, permiten la identificación correcta de especies de Entamoeba,contribuyendoconuna mejor evaluación epidemiológica de estos parásitos y la aplicación adecuada del tratamiento. Recibido: 13-06-2012. Aceptado: 22-11-2012 INTRODUCTION amoebiasis, the latter in some cases in the form of liver abscesses (13). An estimated Entamoeba histolytica is the causal 50 million people suffer from this invasive agent of intestinal amoebiasis, one of the disease worldwide, producing an annual principal causes of mortality in humans death toll of between 40,000 and 100,000 worldwide (1-4). Other Entamoeba species, (14, 15). Although this parasite is distrib- such as E. dispar and E. moshkovskii have uted throughout the world, prevalences ex- also been found in patients with gastroin- ceeding 10% have only been reported from testinal symptoms (5-9). However, there is some developing countries (16). Despite as yet no definitive evidence demonstrating the availability of effective treatments that these two species are pathogenic to against E. histolytica, morbidity and mortal- humans (10-12). ity rates have persisted, suggesting that Clinical features of amebiasis range measures to eliminate or limit the disease from asymptomatic colonization to amoe- are at present largely ineffective. Neverthe- bic dysentery and invasive extra intestinal less, as humans are apparently the only Investigación Clínica 53(4): 2012 Over-diagnosis of E. histolytica en Barcelona, Venezuela 367 hosts, an appropriate control program protocols have been developed, and al- should be able to eradicate the infection though several studies have compared the (17, 18). different methods of species-specific diag- E. histolytica infections are diagnosed noses (25-27), molecular techniques have based on the study of the clinical symptoms, been effective for the successful detection as well as the microscopic examination of se- and differentiation of E. histolytica and E. rial stool samples. The latter is a faster and dispar in clinical samples. Parija and easier procedure, but its sensitivity is lim- Khaimar (7) analyzed 746 stool samples ited and requires an experienced observer to with cysts and trophozoites of E. histolytica, accurately distinguish between pathogenic E. dispar and E. moshkovskii, using PCR to and non-pathogenic species; a serious cause amplify the small rRNA subunit. These au- of error for the correct diagnosis of the dis- thors found a greater prevalence of E. ease. For this reason, direct diagnosis by it- dispar (8.8%) compared to E. histolytica self is considered to be insufficient for the (1.7%). E. histolytica was, in fact, only actu- identification of E. histolytic and should be ally present (as diagnosed by PCR) in 19% complemented by fixation techniques such of the 68 stool samples registered as con- as permanent trichrome staining that per- taining E. histolytica by microscopic exami- mit the intracellular elements to be visual- nation, implying that 81% of suspected in- ized more easily (19,20). Haque et al. (1), fections were wrongly diagnosed and pa- however, argue that trichrome and iron tients were thus unnecessarily treated with hematoxylin staining are not good methods antiamoebic medication. This over-diagno- for the detection of E. histolytica, because sis of E. histolytica, has led to a re-evalua- they do not differentiate between this and tion of the epidemiology of amoebiasis in other non-pathogenic species which are terms of prevalence and morbidity, particu- morphologically identical. larly in geographical areas with high The diagnosis of false negatives of E. endemicity (19,28). histolytica is thus at least partly due to a de- In Venezuela, 6,872,282 cases of diar- lay in sample processing, short analysis rhea, mainly in children, were reported be- time, analyses performed by technicians tween 2007 and 2010. Of these, 574,225 without adequate theoretical and practical were the result of amoebiasis, with Zulia training and a lack of complementary meth- State having the largest number of cases ods that help to improve the visualization of and Anzoátegui and Sucre being the most hematophagous trophozoites (21). False affected states in the eastern region of the positives, on the other hand, occur due to country (29). In addition, changes in the the incorrect identification of non-patho- frequencies of the E. histolytica/E.dispar genic species of amoeba and host cells such complex have been reported, with high as macrophages (22-24). The difficulties of prevalences in different regions (30-33). differentiating E. histolytica from other The application of PCR by Rivero et al. (34) Entamoeba spp. is, in many cases, the rea- in Maracaibo (Western Venezuela), and son why the prevalence of this infection Mora et al. (35), in Cumaná (Eastern Vene- vary so greatly from one region to another zuela), have shown significant differences in (5). the frequencies of infections by species of In order to provide a solution to the Entamoeba between the microscopic and problems of the diagnosis of Entamoeba re- molecular detection. lated infections, rapid and highly sensitive The fact that an important number of Polymerase Chain Reaction (PCR)-based infections
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