Distribution and Differential Diagnosis of Entamoeba Histolytica from Entamoeba Dispar by the PCR-RFLP Method in Central Iran
2003; King Faisal Specialist Hospital and Research Centre; Volume: 23; Issue: 6 Linguagem: Inglês
10.5144/0256-4947.2003.363
ISSN0975-4466
AutoresHossein Hooshyar, M Rezaian, Bahram Kazemi,
Tópico(s)Diagnosis and treatment of tuberculosis
ResumoOriginal ArticlesDistribution and Differential Diagnosis of Entamoeba Histolytica from Entamoeba Dispar by the PCR-RFLP Method in Central Iran Hossein Hooshyar, PhD Mostafa Rezaian, and PhD Bahram KazemiPhD Hossein Hooshyar Correspondence to: Dr. H. Hooshyar, Department of Parasitology, School of Public Health, Tehran University of Medical Sciences, P.O. Box 6446-14155, Tehran, Iran From the Department of Medical Parasitology and Mycology, School of Public Health, Tehran University of Medical Sciences, Tehran, Iran , Mostafa Rezaian From the Department of Medical Parasitology and Mycology, School of Public Health, Tehran University of Medical Sciences, Tehran, Iran , and Bahram Kazemi From the Department of Medical Parasitology and Mycology, School of Public Health, Tehran University of Medical Sciences, Tehran, Iran Published Online:1 Nov 2003https://doi.org/10.5144/0256-4947.2003.363SectionsPDFCite ToolsAdd to favoritesDownload citationTrack citations ShareShare onFacebookTwitterLinked InRedditEmail AboutAbstractBACKGROUNDEntamoeba histolytica and Entamoeba dispar are two morphologically indistinguishable human protozoan parasites that are genetically distinct species. The potential invasive pathogenic Entamoeba histolytica and non-invasive parasite Entamoeba dispar can be differentiated by molecular and other methods. We used polymerase chain reaction (PCR) to determine the ratio of the two species in a population in Tehran and Karaj in central Iran.MATERIALS AND METHODSHuman stool samples (n=12 148) were randomly collected in Tehran and Karaj and examined for E. histolytica/E. dispar cysts with direct and formalin-ether methods. Eighty-seven (0.7%) cases were positive, of which 49 (62.8%) isolates were successfully cultured in Robinson's medium. A pair of oligonucleotide primers designed from sequence data for genomic DNA coding the 30-KD surface antigen of E. histolytica/E. dispar was used to amplify a 374 base-pair (bp) fragment. The electrophoretic pattern of the PCR product digested with Hinfl restriction enzyme was used for differentiation of the two species.RESULTSThe restriction fragment length polymorphism (RFLP) pattern obtained from a standard E. histolytica isolate had two fragments (219 bp and 155 bp), but the standard isolate of E. dispar showed three fragments (155, 152 and 67 bp). Differential diagnosis of 49 isolates of E. histolytica/E. dispar from Tehran and Karaj using PCR-RFLP revealed that 46 (93.9%) were E. dispar while only 2 (4.1%) were E. histolytica. One person (2%) had a mixed infection and showed both patterns.CONCLUSIONThe differential diagnosis of the potentially pathogenic parasite E. histolytica from the non-pathogenic E. dispar is of clinical and epidemiological importance. This study demonstrated that E. dispar is much more prevalent than E. histolytica among the "cyst passers" in Tehran and Karaj in Central Iran.IntroductionEntamoeba histolytica is a cosmopolitan human protozoan parasite responsible for amoebic colitis and extraintestinal amoebiasis. Although 90% of infected individuals are asymptomatic, WHO estimates that every year more than 40 to 50 million cases of invasive amoebiasis occur in the world, resulting in up to 100 000 deaths.1,2 After malaria, amoebiasis is the second most fatal human protozoan parasitic diseases in the world.1Many people infected with E. histolytica are asymptomatic and never develop clinical symptoms.3 What was earlier known as E. histolytica actually comprises two genetically distinct but morphologically identical species, one potentially pathogenic and the other non-pathogenic.4 On the basis of biochemical, immunological and genetic data, a formal re-description of E. histolytica was published in 1993, separating E. histolytica (potential pathogenic) from the harmless commensal, E. dispar.5 Diagnosis and differentiation of the two species is important in therapy and also from an epidemiological point of view. Analysis of restriction fragment length polymorphism (RFLP) and direct sequencing of various genes amplified by polymerase chain reaction (PCR) has revealed 2.2% to 17% differences in the nucleotide sequences of E. histolytica and E. dispar.6 These findings show that PCR and restriction enzyme digestion could be used as a powerful tool for distinguishing E. histolytica from E. dispar.In Iran, there are no accurate data on E. dispar. This study was carried out to determine the ratio of E. histolytica and E. dispar in a population in Tehran and Karaj in central Iran.MATERIALS AND METHODSThis study was carried out from August 2000 to February 2002 in Tehran and Karaj, the two major metropolitan areas of Central Iran. Fecal samples containing cysts or trophozoites of E. histolytica/E. dispar were collected, and after mass cultivation, a 374 base-pair (bp) sequence of genomic DNA that coded for a 30-KD surface antigen (collagen binding protein type 1) was amplified. The two species were differentiated by restriction endonuclease digestion.7,8Human stool samples were randomly collected from different age groups of healthy individuals from various parts of Tehran and Karaj. Fresh stool samples were examined for ova and cysts directly and after formalin-ether concentration methods.9 Those samples with microscopically positive results for E. histolytica/E. dispar cysts were inoculated into Robinson's medium for mass cultivation.10 After three sub-culture trophozoites were harvested by centrifugation at 400 g for 5 minutes, they were washed three times with phosphate buffered saline (pH=7.2). Approximately 2×106 trophozoites were transferred to 1.5 mL microtube and stored at –80°C until DNA extraction.DNA was extracted from trophozoites by phenol/chloroform and precipitated by ethanol according to the Sambrook method11 with some modifications. Briefly, the sediment containing the trophozoite was suspended in 200 μL STE (100 mM NaCl 10 mM Tris and ImM EDTA), 2% sodium dodecyl sulfate (SDS) and 2 μL proteinase K solution (20 mg/mL). The mixture was incubated at 60C for 3 hours and then boiled for 10 minutes. DNA was phenol/chloroform extracted and ethanol precipitated. The DNA was resuspended in 50 μL deionized distilled water and stored at -20C for PCR amplification.One set of oligonucleotide primer HF (Hooshyar forward) (5' AAG AAA TTGATA TTAATG AAT ATA) and HR (Hooshyar reverse) (5' ATC TTC CAA TTC CAT CAT CAT) were designed and used for PCR amplification. These primers amplify a 374-bp sequence of the gene encoding a 30-KD surface antigen of E. histolytica/E. dispar. DNA amplification was achieved in a total volume of 30 microliters in 0.5 mL microtubes. The reaction mixture contained 10 mM Tris-HCl (pH=8.9) (final concentration), 50mM KCl, 1.5 mM MgCl2, 200 nM each of deoxynucleotide triphosphate (dNTP), 20 pmol each of primers and 0.25 μL of Taq DNA polymerase (5U/μL) (GIBCO BRL). Then 0.5-1.0 μL of DNA was added to the reaction mixture and amplified in an automated PCR machine (Eppendorf, Hamburg, Germany, personal). Thirty cycles of denaturation at 94C for 30 seconds (3 minutes for cycle 1), annealing at 54C for 30 seconds and extension at 72C for 30 seconds (5 minutes for cycle 30) were performed. Ten μL of PCR product was electrophoresed on 1.5% agarose gel (containing 1 ng/mL ethidium bromide) and visualized under ultra violet light for specific band identification. Twenty μL of PCR product was digested by Hinfl restriction endonuclease analysis (Boehringer Mannheim, Mannheim, Germany) for 2 hours at 37 C, electrophoresed on 10% Polyacrylamide gel, stained by ethidium bromide and visualized under ultraviolet light.RESULTSA total of 12 148 stool samples were examined. Eighty-seven (0.7%) cases were positive for E. histolytica/E. dispar complex cysts. All isolates were inoculated into Robinson's medium, of which 49 (62.8%) isolates were cultured successfully.A 374-bp fragment of the genomic DNA of 49 isolates of E. histolytica/E. dispar was amplified using HF and HR primers. The PCR amplification products were identical to those of the positive controls [HK9 and 200-NIH strains of E. histolytica and AS2IR strain of E. dispar (zymodeme=l)]. The PCR product was seen only for E. histolytica/E. dispar and no bands or cross-reactivity were amplified from other intestinal protozoa, including Entamoeba coli, Entamoeba hartmani, Giardia lamblia, lodamoeba butschlii, Endolimax nana, Blastocystis hominis and a negative control (without DNA) (Figure 1).Figure 1. 1.5% agarose gel electrophoresis of the PCR product amplified by HF and HR primers. Lane 1, 100-bp DNA ladder. Lane 2, Entamoeba dispar (AS2IR). Lane 3, E. histolytica (200-NIH). Lane 4-9, E. coli, E. hartmani, Giardia lamblia, lodamoeba butschiii, Endolimax nana, Blastocystis hominis, respectively. Lane 10, negative control.Download FigureThe Hinfl restriction endonuclease has been used for differentiation of E. histolytica from E. dispar.(7, 8) When amplified DNA of E. histolytica strain HK9 or 200-NIH was digested with Hinfl, two fragments were detected (219 and 155 bp), whereas the digested PCR product of the E. dispar strain AS2IR showed three fragments (155, 152, 67 bp), of which 155 and 152 bp fragments were overlapped (Figure 2).Figure 2. 10% polycrylamide gel electrophoresis of Hinfl-digested PCR products. Lane 1, undigested product (374 bp). Lane 2, 100-bp DNA ladder. Lane 3, E histolytica (200-NIH). Lane 4, E dispar (AS2IR). Lane 5-8, clinical samples.Download FigureRFLP analysis of amplified genomic DNA of 49 isolates obtained from infected individuals in Tehran and Karaj showed the following findings: two persons had E. histolytica (4.1%) and 46 (93.9%) individuals were positive for the E. dispar pattern. In addition, one person (2%) was positive for both E. histolytica and E. dispar patterns (Figure 2).DISCUSSIONE. dispar is morphologically indistinguishable from potential pathogenic E. histolytica, but is not able to produce clinical symptoms. These two species could be differentiated by methods such as a specific DNA probe,12 PCR and RFLP,13 monoclonal antibody2,14 and analysis of isoenzyme typing,15 all of which are expensive and time-consuming. Comparison of methods showed that PCR could be useful as a reference test for Entamoeba sensitive differentiation of both species.16 A WHO Expert Consultation on Amoebiasis stressed the need for development and improvement of simple methods for specific diagnosisof E. histolytica and E. dispar in clinical and epidemiological studies.1 It is now clear that even in countries in which amoebiasis is endemic, such as southern Africa and India, E. dispar is the dominant species, and the ratio of E. dispar to E. histolytica is approximately 10:1.3 In Cote d'Ivoire this ratio is as much as 46:1.17 So in most cases antiparasitic therapy is not necessary.In Iran, a number of epidemiological studies have been carried out on E. histolytica infection using routine stool examination techniques. However, there is no accurate data on the exact species identity of the parasites. In Iran, the prevalence rate of E. histolytica among healthy individuals ranges from >1% to 30%.18 However, since it is impossible to differentiate E. histolytica from E. dispar by routine stool examination methods, we determined the ratio of E. histolytica to E. dispar by PCR. This is the first report of E. dispar distribution in Iran in which the two distinct species have been considered. This study showed that E. histolytica is rare in Tehran and Karaj and E. dispar (96.9%) is the predominant species in this region. This is an important finding because E. dispar infection does not need to be treated. It is interesting to note that in this area most cases reported as "E. histolytica cyst passer" are actually infected with E. dispar. According to the WHO expert groups it is better to identify and treat E. histolytica specifically,1 but this is not possible until a simple and cheap method for detection and differentiation of the two species in endemic areas is available.We found a low incidence of E. histolytica as well as mixed infection with both E. histolytica and E. dispar in Tehran and Karaj. These findings are consistent with findings in countries such as the Netherlands,19 Canada16 and the Philippines.20 In an epidemiological study in Turkey all of 73 isolates of E. histolytica/E. dispar that were differentiated using colorimetric PCR were E. dispar.21 However, E. histolytica has been reported in other areas such as Bangladesh13 and Mexico as the predominant species and a large number of mixed infections have been reported.22 Today, it is accepted that in areas where invasive amoebiasis is common, E. dispar is also a prevalent species.23We believe that E. dispar is the predominant species in the central regions of Iran. A high percentage of cases reported as E. histolytica are actually E. dispar. Because previous studies on the prevalence and incidence of E. histolytica in Iran were not based on specific differentiation of the two species, a large-scale study of infected individuals in different areas of Iran is necessary to estimate the true incidence and prevalence of each species.ARTICLE REFERENCES:1. World Health Organization. "Entamoeba taxonomy." Bull World Health Organ.. 1997; 75:291-292. Google Scholar2. Petri WA, Haque R, Lyerly D, Vines RR. "Estimating the impact of amoebiasis on health." Parasitol Today.. 2000; 16:320-321. Google Scholar3. Clark CG. "Entamoeba dispar, an organism reborn." Trans R Soc Trop Med Hyg.. 1998; 92:361-364. Google Scholar4. Haque R, Ali IK, Akther S, Petri WA. "Comparison of PCR, isoenzyme analysis and antigen detection for diagnosis of Entamoeba histolytica infection." J Clin Microbiol.. 1998; 36:449-452. Google Scholar5. Diamond LS, Clark CG. 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Zaki M, Meelu P, Sun W, Clark CG. "Simultaneous differentiation and typing of E. histolytica and E." dispar. J Clin Microbiol.. 2002; 40:1271-1276. Google Scholar Previous article Next article FiguresReferencesRelatedDetails Volume 23, Issue 6November-December 2003 Metrics History Accepted1 March 2003Published online1 November 2003 KeywordsEntamoeba histolyticaEntamoeba disparPCR-RFLPIranACKNOWLEDGEMENTSThe authors would like to thank Ms. Farnia and Ms. Babaie in the School of Public Health, Tehran University of Medical Sciences for providing the axenic standard strains used in this study. We also thank Dr. M. Fasihi and Dr. S. Solaymani-Mohammadi for reading the manuscript.InformationCopyright © 2003, Annals of Saudi MedicinePDF download
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