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

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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: , diagnosis, PCR, E. histolytica.

Abstract. The aim of this study was to identify the presence of 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 , 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 by Entamoeba spp. has been re-

Vol. 53(4): 353 - 364, 2012 368 Rodulfo y col. ported in Venezuela, means that more ap- thorized the participation of their child or propriate, sensitive and specific methods children and the stools had been processed that allow for the correct detection and dif- by microscopy, l g of fecal matter was re- ferentiation of species within this genus moved from each sample and placed in a should be implemented. We thus undertook 1.5 mL sterile Eppendorf tube with 500 µL this investigation in order to assess the real phosphate buffered saline (PBS) 1X. The frequencies of E. histolytica and E. dispar in tubes were then transported on ice to the stool samples diagnosed as positive for E. Molecular Genetics Laboratory, IIBCA- histolytica and/or E. dispar by conventional UDO, where they were stored at –20°C until methods in patients from Barcelona, Vene- analysis by PCR. zuela. DNA extraction METHODOLOGY Genomic DNA was extracted from the 150 stool samples using the Promega Wiz- Study population ard® Genomic DNA purification Kit, em- A total of 150 stool samples collected ploying the protocol suggested by the man- from children aged 0-10 of both sexes, be- ufacturer with the following modifications: tween January and August 2009, who at- 200 µL of each of the fecal samples pre- tended the Pediatric Department “Dr. served in PBS were placed in a 1.5 mL Rafael Tobías Guevara” at the Dr. “Luis Eppendorf tube, centrifuged at 14 000 g for Razetti” Hospital, Barcelona, Anzoátegui 3 min at 18°C and the supernatant dis- State, were diagnosed as microscopically carded. Then 400 µL nuclei lysis solution positive for Entamoeba histolytica, when was added and the mixture homogenized by they were observed using wet preparations pipetting the sediment. Cyst walls were with 0.85% physiological saline solution and fractured by sonication, using a high inten- Lugol’s solution. The physical characteris- sity sonicator (Autotone Ultrasonic) set at tics of the samples such as: appearance, the minimum speed for 2 sec and repeated consistency, color, odor, pH, presence or three times, with the samples held on ice. absence of mucus, blood and/or adult 10 µL proteinase K was then added. Sam- worms, were noted as well as the signs and ples were incubated for 2 hours at 65°C and symptoms of the children at the time of the then left to cool for 5 min at room temper- collection of the sample (fever, diarrhea, ature. The extraction procedure was then abdominal pain, among others). In addi- continued using the protocol recommended tion, intestinal worms and protozoa were by the kit and the DNA obtained stored at detected and identified microscopically in –20°C, until its subsequent amplification by each of the samples. PCR. The consent of the legal representa- tives of the children who participated in the (PCR) polymerase chain reaction study was sought in each case after inform- The Nested PCR procedure has been ing them about the aims of the research. proposed by the International Centre for All of the methods used were previously ap- Diarrhoeal Disease Research, Dhaka, Ban- proved by the Bioethics and Biosafety Com- gladesh (ICDDR, B) (36), as well as the mittee at the Instituto de Investigaciones Jawaharlal Institute of Postgraduate Medi- en Biomedicina y Ciencias Aplicadas, Uni- cal Education and Research (JIPMER), versidad de Oriente (IIBCAUDO), Cumaná, Puducherry, India (7) as the optimal Venezuela. Once the representative had au- method for the detection and differentia-

Investigación Clínica 53(4): 2012 Over-diagnosis of E. histolytica en Barcelona, Venezuela 369 tion of E. histolytica and E. dispar, using with the Chi-square (c2) test (37, 38), us- the 16S rRNA-like gene sequence as the ing version 11.5 of the SPSS statistical genomic target. package. Nested PCR was performed by first am- plifying a fragment specific to the RESULTS Entamoeba genus (E1-E2 primers 898 bp) and afterwards the fragments specific to E. E. histolytic was found in 13.2% (150) histolytica and E. dispar (439 bp, and174 of the 1,141 stool samples analyzed micro- bp, respectively) following the protocol de- scopically by bioanalysts in the Pediatric scribed by Parija and Khairnar (7). A culti- Department Laboratory at the “Dr. Luis vated strain of E. histolytica; IULA-0593:2 Razetti" Hospital, Barcelona, Anzoátegui (NER), donated by the Immunology Insti- State, of which 79.3% contained cystic tute, Universidad de los Andes (ULA), forms and trofozoites of E. histolytica (Ta- Mérida, Venezuela; and a positive sample ble I). In addition, in 19.3% of the samples, for E. dispar, UDO402 strain (35), were contained forms of other intestinal proto- used as positive controls in each PCR run. zoa such as Giardia duodenalis, Blastocystis The amplified products were separated by hominis, E. coli and Chilomastix mesnili; electrophoresis on a 2% agarose gel (EC330 and in 1.4%, co-infections with the eggs of Primo Minicell Gel Electrophoresis System) intestinal helminthes, such as Trichuris in Tris-boric acid-EDTA (TBE) buffer 1X at trichiura and Hymenolepis nana, were found 80 V for 45 min, stained with ethidium bro- (Table I), demonstrating a high prevalence mide (0.5 µg/mL) and visualized in a ultra- of infections caused by intestinal protozoa violet transilluminator. The molecular in the samples analyzed. However, it must marker used was the Axygen100 pb ladder. be emphasized that these samples were not concentrated as part of the clinical diagno- Statistical analysis sis, thus the frequency of helminths and The results obtained are shown in the other intestinal protozoa observed may tables and figures. Associations between have been underestimated. the Entamoeba species assessed in this A total of 98.7% of the children study with the age, sex and symptoms of showed symptoms related to the infection, the patients and the macroscopic charac- with acute diarrhea being the most preva- teristics of the fecal material, were tested lent symptom (68%), followed by fever

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

Symptom E. histolytica E. histolytica, E. histolytica, E. histolytica, Total G. duodenalis E. coli T. trichiura B. hominis C. mesnili H. nana Acute diarrhea 80 12 8 2 102 Fever 19 3 2 0 24 Abdominal pain 16 1 2 0 19 Dysentery 20103 Asymptomatic 20002 Total 119 16 13 2 150

Vol. 53(4): 353 - 364, 2012 370 Rodulfo y col.

(16%) and abdominal pain (12.7%) The first round of amplification of the (Table I). When conventional microscopy nested PCR, corresponding to the 898 bp was used as the diagnostic method, E. fragment specific for Entamoeba spp., al- histolytica was identified as single infection lowed us to detect a 28% (42/150) infection in 79.3% of symptomatic and asymptomatic rate by any of the Entamoeba species. The children and in only 12% of samples they second round of amplification produced spe- were accompanied by other protozoa and cies specific fragments in all of these sam- pathogenic intestinal helminths. In 8.7% of ples, of which 29 (19.3%) were identified as samples, E. histolytica was found causing in- E. histolytica,6(4%)asE. dispar,and7 fections together with commensal protozoa (4.7%) as mixed infections by E. such as E. coli and C. mesnili (Table I). histolytica/E.dispar (Eh/Ed)(Figs.1and2).

Fig. 1. Representative gels for the second round of the nested PCR showing the presence of E. his- tolytica as diagnosed from stool samples collected from the studied children. M: 100 bp mole- cular weight marker (Axygen). C+: E. histolytica NER strain positive control: 439 bp. Evalua- ted 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.

Investigación Clínica 53(4): 2012 Over-diagnosis of E. histolytica en Barcelona, Venezuela 371

The PCR procedure applied to the 150 TABLE II stool samples microscopically diagnosed as FREQUENCY OF THE SYMPTOMS SHOWN BY positive for E. histolytica showed that mi- THE CHILDREN UNDER STUDY FOR EACH OF croscopic analysis over-diagnosed the inci- THE INFECTING AGENTS AS DIAGNOSED BY PCR dence of amebiasis by 72% (108/150). As already mentioned above, PCR showed that Symptoms Eh Ed M only 42 of the 150 samples were, in fact, Acute diarrhea 18 4 5 positive for the Entamoeba species diag- nosed in this investigation. Fever 4 2 1 Furthermore, PCR revealed that in Abdominal pain 6 0 1 89.7% (26/29) of infections, E. histolytica Dysentery 0 0 0 was the only parasite involved, whereas in 10.3% (3) of the cases there was a co-infec- Asymptomatics 1 0 0 tion with E. coli and C. mesnili. In those in- Total 29 6 7 fections caused by E. dispar and mixed Eh: Entamoeba histolytica. Ed: Entamoeba dispar. Eh/Ed infections no other parasites were M: mixed Eh/Ed infection. found, except for a mixed infection with E. coli and C. mesnili. It should be noted that dispar and mixed Eh/Ed infections, and E. histolytica was not detected in samples 33.3%of children infected by E. dispar also where it had been microscopically diag- suffered fever. These observations thus nosed as co-infecting with other protozoa show that both E. histolytica and E. dispar and pathogenic intestinal helminthes. This may be found in the same group of individu- could be due to the difficulty of differentiat- als with intestinal symptoms. ing Entamoeba spp. from host cells when We could not find any species specific using SSF and Lugol´s solution as the stain- pattern as regards stool consistency when ing agents during parasitological diagnosis. we macroscopically examined the PCR posi- Regarding the symptoms associated tive stool samples (Table III), as both soft with the infections (Table II), diarrhea was andliquidstoolswereinfectedbyeitherspe- also the most common symptom of infec- cies. Mixed infections did, however, predomi- tion by E. histolytica (62.1%), followed by nate in liquid samples (71.4%). Similarly, abdominal pain (20.7%) and fever (13.8%). the presence of blood was not necessarily in- Diarrhea was also present in 66.6% of all E. dicative of pathogenic species, although it

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

PCR Results N Consistency Mucus Blood Liquid Soft E. histolytica 29 10 19 28 17 E. dispar 63363 E. histolytica/E. 75264 dispar Negative 108 47 61 0 0 Total 150 65 85 40 24

Vol. 53(4): 353 - 364, 2012 372 Rodulfo y col. was statistically associated with E. histolytica quent (80.2%) in the 6-16 year-old age- (c2 = 4.753; P < 0.05) (Table IV). Lastly, group, with the most common species be- mucus was found in the fecal samples of ing, Endolimax nana, E. coli, G. duodenalis most of the E. histolytica, E. dispar and and the Eh/Ed complex; this last with a mixed Eh/Ed infections (Table III). prevalence of 11.3%. This confirms the presence and range of these micro-organ- DISCUSSION isms and indicates that an understanding of their distribution in areas of transmission In general, infections caused by intes- of pathogenic protozoa could aid the devel- tinal protozoa predominated in the children opment of disease control programs that examined. In this regard, authors such as combine treatment with prevention. Ouattara et al. (39) indicate that poly- With respect to the prevalence of E. by intestinal protozoa is fre- histolytica in Venezuela, large variations in

TABLE IV SUMMARY OF THE RESULTS OF THE c2 ANALYSIS OF THE ASSOCIATION OF THE MACROSCOPIC CHARACTERISTICS OF THE STOOL SAMPLES, AGE, SEX AND GASTROINTESTINAL SYMPTOMS OF THE CHILDREN WITH THE PRESENCE OF E. histolytica AND E. dispar AS IDENTIFIED BY PCR

Species Variable c2 p Sex 0.068 0.794 Age 1.071 0.586 Diarrhea 1.033 0.309 Fever 0.000 1.000 Consistency 0.054 0.973 E. histolytica Mucus 0.220 0.639 Blood 4.753 0.029* pH 1.397 0.237 Leucocytes 0.070 0.792 Red bloodcells 0.130 0.718 Sex 0.004 0.950 Age 0.274 0.872 Diarrhea 0.273 0.601 Fever 0.421 0.515 Consistency 2.410 0.300 E. dispar Mucus 1.358 0.244 Blood 0.727 0.394 pH 0.007 0.935 Leucocytes 1.125 0.289 Red bloodcells 0.000 0.997 *Significantly associated variable, P < 0.05.

Investigación Clínica 53(4): 2012 Over-diagnosis of E. histolytica en Barcelona, Venezuela 373 different regions of the country have been polymorphonuclears (PMN) and cysts, lead- reported. In the West, a prevalence of 4% ing to the incorrect identification of amoe- for the E. histolytica/E. dispa rcomplex was bas, is well known (19, 22, 23). Thus, the detected by parasitological analysis (33). A current recommendation when species spe- study of the Yukpa ethnicity in rural areas cific diagnosis is not possible is to report: of Zulia state, showed a prevalence of 21.9% “E. histolytica/E. dispar/E. moshkovskii”in E. histolytica infections in children aged order to describe the presence of species 0-14 (32). In addition, Rivero et al. (34), with identical morphologies in stool sam- obtained a prevalence of 10.8% E. ples. Researchers and technicians are also histolytica infections in Maracaibo, Zulia encouraged to use new technologies wher- state using PCR, compared with that of ever possible in order to elucidate the true 20.6% mixed E. histolytica/E. dispa rinfec- epidemiology and pathogenesis of tions diagnosed by parasitological analysis. Entamoeba spp., including the least studied These researchers found no significant asso- E. moshkovskii (41). ciations between infection by these species The fact that 108 samples were diag- and age and/or sex. nosed by conventional microscopy as posi- In Bolívar state (southeast Venezuela), tive for E. histolytica, but were found to be Devera (40) reported the absence of this in- negative for this species by PCR, may be ex- testinal protozoa after microscopic analysis. plained by the presence of intestinal amoe- However, Mora et al. (35) found a preva- bas morphologically similar to E. histolytica lence of 5.4% for E. histolytica and 3.5% for such as E. hartmanni and E. polecki.This E. dispar infections using molecular detec- last, although it is a parasite of monkeys and tion by PCR in individuals with gastrointes- pigs, and is uncommon in humans, has been tinal symptoms in Cumaná, Sucre state. Ac- reported from the latter on eight occasions cording to recent reports there is a higher in Venezuela (42-45). E. hartmanni,onthe prevalence of E. histolytica in Sucre state, other hand, has often been reported from compared to other states. This coincides western regions of Venezuela since 1976, us- with our results for Barcelona, Anzoátegui ing staining and concentration techniques state, showing higher frequencies in North- (31, 45, 46-50). All these studies have eastern Venezuela, compared to the South- pointed out that E hartmanni may be less eastern area. frequently detected in fresh stools or con- Our study agrees with that of Mora et centrated samples due to its small size, or al. (35), in that both indicate an important because its morphological features make it over-diagnosis of pathogenic species by indistinguishable from other Entamoeba spe- parasitological methods. They reported cies, thus hindering a specific diagnosis. 51.2% false positives from conventional mi- The macroscopic characteristics of the croscopic analysis. On the contrary, Rivero stool are important to indicate the correct et al. (34) found that under-diagnosed by diagnostic methodology to use in the search 10.6% by microscopic examination: 42 posi- for intestinal pathogens. In this study, how- tive samples were detected by this method ever, the characteristics of PCR-positive compared to 47 positives by PCR, however samples (consistency and presence of mu- only 22 (10.78%) of these were identified as cus) were not reliable indicators of the pres- E. histolytica. ence of the different Entamoeba spp. Never- The tendency of microscopic analysis theless, the presence of blood, although not to produce false results due to confusion exclusive to one pathogenic species, did between macrophages and trofozoites, and show a statistically significant association

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