Aim: The present study was performed in order to differentiate E. histolytica and E. dispar in children from Gorgan city, using a PCR method. Background: Differential detection of two morphologically indistinguishable protozoan parasites Entamoeba histolytica and E. dispar has a great clinical and epidemiological importance because of potential invasive pathogenic E. histolytica and non-invasive parasite E. dispar. Patients and methods: One hundred and five dysentery samples were collected from children hospitalized in Taleghani hospital in Gorgan city. The fecal specimens were examined by light microscopy (10X then 40X) to distinguish Entamoeba complex. A single round PCR amplifying partial small-subunit rRNA gene was performed on positive microscopy samples to differentiate E. histolytica/ E. dispar and E. moshkovskii from each other. Results: Twenty-five specimens (23.8%) were positive for Enramoeba complex in direct microscopic examination. PCR using positive controls indicated E. histolytica and E. dispar in two (2/25, 8%) and three (3/25, 12%) samples, respectively. Conclusion: There is a warrant to performing molecular diagnosis for stool examination at least in hospitalized children in order to prevent incorrect reports from laboratories and consequently mistreating by physicians. © 2014 RIGLD, Research Institute for Gastroenterology and Liver Diseases

Cheraghali, F.

Maghsoodloorad, F.S.

Nazemalhosseini-Mojarad, E.

Sharbatkhori, M.

Taherkhani, H.

Vakili, M.

English

PubMed

Golestan University of Medical Sciences Repository

                                         1              Discrimination of Entamoeba Spp. in children with dysentery Mitra Sharbatkhori1,2, Ehsan Nazemalhosseini-Mojarad3, Fatemeh Cheraghali4, Fatemeh SoghraMaghsoodloorad2, Heshmatolla Taherkhani2, Mohammadali Vakili5 1Infectious Diseases Research Center, Golestan University of Medical Sciences, Gorgan, Iran2Department of Parasitology and Mycology, School of Medicine, Golestan University of Medical Sciences, Gorgan, Iran 3Gastroenterology and Liver Disease Research center, Shahid Beheshti University of Medical Sciences, Tehran, Iran 4Department of Pediatric Diseases, Golestan University of Medical Sciences, Gorgan, Iran 5Department of Community Medicine, School of Medicine, Golestan University of Medical Sciences, Gorgan, Iran  ABSTRACTAim: The present study was performed in order to differentiate E. histolytica and E. dispar in children from Gorgan city,using a PCR method.Background: Differential detection of two morphologically indistinguishable protozoan parasites Entamoeba histolytica and E. dispar has a great clinical and epidemiological importance because of potential invasive pathogenic E. histolytica and non-invasive parasite E. dispar. Patients and methods: One hundred and five dysentery samples were collected from children hospitalized in Taleghanihospital in Gorgan city. The fecal specimens were examined by light microscopy (10X then 40X) to distinguish Entamoeba complex. A single round PCR amplifying partial small-subunit rRNA gene was performed on positive microscopy samples to differentiate E. histolytica/ E. dispar and E. moshkovskii from each other. Results: Twenty-five specimens (23.8%) were positive for Enramoeba complex in direct microscopic examination. PCR using positive controls indicated E. histolytica and E. dispar in two (2/25, 8%) and three (3/25, 12%) samples, respectively.  Conclusion: There is a warrant to performing molecular diagnosis for stool examination at least in hospitalized children in order to prevent incorrect reports from laboratories and consequently mistreating by physicians.Keywords: Entamoeba histolytica, Entamoeba dispar, children, dysentery, PCR, Gorgan, Iran. (Please cite as: Sharbatkhori M, Nazemalhosseini-Mojarad E, Cheraghali F, Maghsoodloorad FS, Taherkhani H,Vakili MA. Discrimination of Entamoeba Spp. in children with dysentery. Gastroenterol Hepatol Bed Bench2014;7(3):164-167).Introduction  Amoebiasis is still mentioned as one of the main health problems in tropical and subtropical regions (1). The true prevalence of infection caused byEntamoeba histolytica is unknown for most areas of the world (2). Gastroenterology and Hepatology From Bed to Bench. ©2014 RIGLD, Research Institute for Gastroenterology and Liver DiseasesORIGINAL ARTICLEReceived: 19 March 2014 Accepted: 24 May 2014 Reprint or Correspondence: Mirta Sharbatkhori, PhD. Department of Parasitology and Mycology, GolestanUniversity of Medical Sciences, Gorgan, Iran.E-mail: msharbatkhori@yahoo.comE. histolytica causes widespread mortality and morbidity worldwide through diarrheal disease and abscess establishment in parenchymal tissuessuch as liver, lung, and brain (3). In contrast, other amoebae that infect humans include E. dispar, E.moshkovskii, E. coli, E. hartmanni, and Endolimax nana, have been considered nonpathogenic (4). E. histolytica, E. dispar, and E. moshkovskii aremorphologically indistinguishable but are different biochemically and genetically. Although E. histolytica is recognized as a pathogen, the ability Gastroenterol Hepatol Bed Bench 2014;7(3):164-167                                                                                                                               Sharbatkhori M. et al  165 of the other two species to cause disease is unclear(5). It is also worthy to note that until recently the differentiation of E. histolytica from the non-pathogenic amebic species was not possible (6, 7).The epidemiology of E. histolytica in Iran is poorly understood. Fortunately, several studies over the last decade have begun to evaluate the prevalence or incidence of E. histolytica in specific populations of Iran, these studies were utilized different molecular methods in order to differentiate the non-pathogenic E. dispar or E. moshkovskii from the pathogenic E. histolytica (8­16). In this study, we used the well-characterizeddiagnostic tests (single round PCR) to examine the prevalence of E. histolytica, E. dispar and E. moshkovskii infections in children with dysenteryin Golestan province, Iran. Patients and Methods From January 2010 to September 2012, 105dysentery samples were collected from childrenhospitalized in Taleghani hospital in Gorgan city, the capital of Golestan Province, located innorthern Iran and south east of Caspian sea. Socio­demographic and clinical data were collected from the child’s parents and medical records. Stool specimens were screened microscopicallyusing direct slide smear for the presence ofEntamoeba spp. (13). Genomic DNA was extracted directly from stool specimens weremicroscopically positive by using a QIAamp® DNA Stool Kit (QIAGEN) according to the manufacturer's instructions. The extracted DNAwas stored at -20°C until PCR amplification. A single–round PCR reaction and primers setsamplifying partial small-subunit rRNA gene were used as described previously (13, 17, 18). The sequence of the forward primer used was conserved in all three Entamoeba spp., but the reverse primers were specific for apiece. Theexpected PCR products from E. histolytica, E. dispar and E. moshkovskii were 166 bp, 752 bp,and 580 bp, respectively (17). Amplification of each species–specific DNA fragment started with an initial denaturation at 94°C for 3 min, followed by 30 cycles of 94°C for 1 min,58°C for 1 min, and 72°C for 1 min, with a final extension at 72°C for 7 min (14). PCR products werevisualized with ethidium bromide staining after electrophoresis on 1.5% agarose gels. DNA isolatedfrom axenically grown E. histolytica KU2, E. disparAS 16 IR and E. moshkovskii Laredo (ATCCaccession no. 300 42) (13) were used as positive controls. Medical Research Ethics Committee of Golestan University of Medical Sciences approved the study.1 2 3 4 5 6 7 8 N M 752 bp 580 bp 166 bp Figure 1. Agarose gel electrophoresis of Entamoebaspecies using single–round PCR. Lanes 2-3: E.histolytica – positive isolates; Lane 5-7: E. dispar – positive isolates; Lanes 1, 4 and 8: E. histolytica , E. dispar  and E. moshkovskii positive controls, respectively. N: Negative control. M: 100 bp ladder DNA size marker Results Out of the 105 children with dysentery, 25(23.8%) cases were infected with E. histolytica/E.dispar/E. moshkovskii complex. After DNA extraction, the single-round PCR was carried out to differentiate the Entamoeba spp. of 25 samples that Gastroenterol Hepatol Bed Bench 2014;7(3):164-167                                                        166 Discrimination of Entamoeba Spp. in children with dysenterywere microscopically positive. Three (12%) E. dispar and 2 (8%) E. histolytica were detected by PCR (Figure 1). Infection of E. moshkovskii was not observed in this study. Amplification produced fragments of 166bp,752bp and 580bp corresponding to the expected products from E. histolytica, E. dispar and E.moshkovskii, respectively. Discussion Infection with E. histolytica is a severe healthproblem in many tropical and subtropical areas of the world, especially in developing countries such as Iran (19, 20). It is now known that most of human cases ofinfection with E. histolytica/E. dispar are actually E. dispar. E. dispar is non-pathogenic, and requires no treatment. Because of this, differential diagnosis of the pathogen E. histolytica from the commensally E. dispar is of the utmostimportance (21). Most epidemiological studies for E. histolyticainfection were performed before the acceptance ofE. histolytica/E. dispar and E. moshkovskii as distinct species. There is a clear need to perform new epidemiological studies to distinguish these three species of Entamoeba and to find trueprevalence of E. histolytica species (14).The results of this study clearly show that, microscopy is not a sensitive and reliable technique for diagnosing amebic dysentery as well as differentiation of E. histolytica from E. disparand E. moshkovskii, because most amebicinfections in this population were due to other nonpathogenic Entamoeba species (17). In a previous study in Gonbad city 16 (69.5%) from 23microscopic positive isolates for Entamoebaspecies were amplified in PCR as E. dispar, none of the isolates (0%) revealed as E. histolytica andthe seven isolates were not amplified in PCR. The higher prevalence of E. dispar is in concordance with our study (10). The 20 negative PCR isolates (30.43%) with both two sets of E. histolytica and E. dispar primers in this study might be due tolack of enough DNA template, PCR error or misdiagnosis with some other Entamoeba species.However, this conjecture should be confirmed bythe further development of molecular diagnosis forother nonpathogenic Entamoeba speciescommonly found in humans, such as E. coli and E.hartmanni (17).While the isolates were achieved typically from patients who suffered from dysentery, we found no E. histolytica among the isolates and observedno correlation between the presences ofEntamoeba spp. and clinical symptoms, such as abdominal pain, diarrhea or nausea. It may be thatthe clinical symptoms of some patients were due to viral or bacterial pathogens not detected by the tests that had been run as in a valid study,Kermani et al. showed that Enteroaggregative Escherichia coli was the most prevalent pathogen in both persistent and acute diarrhea children admitted to a pediatric hospital (22).In conclusion, the finding of our study emphasis to perform molecular diagnosis for stool examination at least in hospitalized children in order to prevent incorrect results from medicallaboratories and consequently mistreating byphysicians. Acknowledgment The authors would like to thank Fatemeh Malek, Zeinab Golalikhani, Hamid Mirkarimi and Akbar Mirbazel in the Taleghani hospital who helped to collect the samples. This work was financially supported by Golestan University of Medical sciences, project code: 8912240184.References 1. Tanyuksel M, Petri W A. Laboratory Diagnosis of Amebiasis. Clin Microbiol Rev 2003;16:713-729.2. Stauffer W, Ravdin J. Entamoeba histolytica: anupdate. Curr Opin Infect Dis 2003;16:479-85. Gastroenterol Hepatol Bed Bench 2014;7(3):164-167                                                                                          Sharbatkhori M. et al  167 3. Irusen E M, Jackson T F H G, Simjee A E.Asymptomatic Intestinal Colonization by Pathogenic Entamoeba histolytica in Amebic Liver Abscess: Prevalence, Response to Therapy, and Pathogenic Potential. Clin Infect Dis 1992;14:889-93. 4. Blessmann J, Ali IK, Nu PA, Dinh BT, Viet TQ,Van AL, et al. Longitudinal study of intestinalEntamoeba histolytica infections in asymptomatic adult carriers. J Clin Microbiol 2003;41:4745-50. 5. Fotedar R, Stark D, Beebe N, Marriott D, Ellis J,Harkness J. PCR detection of Entamoeba histolytica, Entamoeba dispar, and Entamoeba moshkovskii instool samples from Sydney, Australia. J Clin Microbiol 2007;45:1035–37. 6. Ali IKM, Clark CG, Petri Jr W A. Molecular epidemiology of amebiasis. Infect Genet Evol 2008;8:698-707. 7. Espinosa-Cantellano M, Martínez-Palomo A. Pathogenesis of Intestinal Amebiasis: From Moleculesto Disease. Clin Microbiol Rev 2000;13:318-31.8. Hooshyar H, Rezaian M, Kazemi B, Jeddi-TehraniM, Solaymani-Mohammadi S. The distribution ofEntamoeba histolytica and Entamoeba dispar in northern, central, and southern Iran. Parasitol Res 2004;94:96-100. 9. Solaymani-Mohammadi S, Rezaian M, Babaei Z,Rajabpour A, Meamar A R, Pourbabai A A, et al. Comparison of a Stool Antigen Detection Kit and PCRfor Diagnosis of Entamoeba histolytica and Entamoeba dispar Infections in Asymptomatic Cyst Passers in Iran. J Clin Microbiol 2006;44:2258-61.10. Nazemalhosseini Mojarad E, Haghighi A, Azimi RadM, Mesgarian F, Rostami Nejad M, Zali M. Prevalenceof Entamoeba histolytica and Entamoeba dispar inGonbad city, Iran. Iran J Parasitol 2007;2:48-52.11. Nazemalhosseini Mojarad E, Haghighi A, Kazemi B, Rostami Nejad M, Abadi A, Zali M. 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Discrimination of Entamoeba spp. in children with dysentery

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Discrimination of Entamoeba spp. in children with dysentery

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