Original article
Detection and differentiation of two morphologically identical species of Entamoeba Abeba Gebertsadik1, Amha Kebede1, Mekonnen Mezemer2, Geremew Tasew1 Abstract Background: Infection with Entamoeba histolytica is a worldwide public health problem. Diagnosis of this parasite by conventional microscopy is almost impossible, because it is morphologically indistinguishable from E. dispar, which is non pathogenic. Objective: To detect and differentiate E. histolytica from E. dispar by PCR technique and to determine the prevalence of trophozoites and cysts of E. histolytica /E. dispar by microscopy of fresh stool specimen among out patients of Jimma University Hospital to provide baseline data for further study. Method: A cross-sectional study was conducted from Feb. 24 – Mar. 23, 2003 in Jimma University Hospital among patients with clinical signs and symptoms of intestinal complaints. The stool samples were examined microscopically and by PCR and Solution Hybridization Enzyme Linked Immuno assay (SHELA). Results: Out of 228 stool specimens collected, 30(13.2%) E. histolytica/E. dispar were observed by microscopic examination of which 6(2.6%) were trophozoite and 24 (10.5%) were cysts. From the total 30(13.2%) microscopically observed E. histolytica /E. dispar, PCR-SHELA detected 28(12.3%) E.dispar while no E.histolytica DNA was found. Infections with one or more intestinal parasites were common, 151 (66.2 %). By far the most common intestinal parasitic infection was Ascaris lumbricoides, 87 (38.2%) followed by Trichuria trichiura, 53(23.3%). Conclusion: Infection with E. histolytica was not found in all the stool samples examined. Neither the trophozoites nor the quadrinucleated cysts reported by microscopy were those of E. histolytica. Our finding suggests that among the patients studied, the commonly reported trophozoites and cysts belong to the non-pathogenic E. dispar, which requires no treatment at all. Therefore, the commonly reported complaints of diarrhea require alternative explanation since the routine diagnostic microscopy of amebiasis is unsatisfactory. [Ethiop.J.Health Dev. 2004;18(2):121-124] Introduction Entamoeba histolytica is an enteric protozoan parasite that exists in either trophozoite or cyst form. The trophozoite is the motile form that multiplies by binary fission and differentiates to cyst, the resistant form responsible for the transmission of infection. Cysts are excreted in stools and may be ingested by a new host via contaminated food or water (1). E. histolytica is capable of invading the intestinal mucosa and may spread to other extraintestinal organs, mainly the liver and rarely the lung, brain and kidney. Thus E .histolytica is unique among the intestinal amebae, because it is able to invade tissue and clinical presentation may range from an asymptomatic infection to a disseminated fatal disease. Incubation period may vary from a few days to months depending on the area of endemicity (2). Recent studies show that there are two morphologically identical species of ameba (3-5). The first species is E. histolytica which is pathogenic and the second is E. dispar which is non pathogenic. In a recent study on the epidemiology of infections with intestinal parasites and HIV in Ethiopia, E. histolytica / E. dispar was among the most common parasitic infection with a prevalence of
1
25% (6). In a previous study done in Wonji, central Ethiopia, 29 cultured amebic isolates from 123 study participants revealed 27 isolates with E. dispar zymodemes and only 2 isolates with an E. histolytica zymodeme (4). The prevalence of the pathogenic E.histolytica and the non-pathogenic E.dispar is not fully documented in Ethiopia, which leads to unnecessary treatment. This study tries to identify and differentiate E. histolytica and E.dispar by PCR technique among patients attending the outpatient department in Jimma University Hospital due to intestinal disease complaints. Materials and Methods Microscopy A cross-sectional study was conducted on 228 stool samples collected from patients who had complaints for intestinal parasites from Feb. 24 – Mar. 23,2003 in Jimma town which is located 335 km south west of Addis Ababa, the capital city of Ethiopia. Stool was collected with a clean and labeled container and patients were asked and instructed on how to bring approximately 3 g of stool, which was enough for direct saline and iodine as well as formol- ether concentration
Ethiopian Health and Nutrition Research Institute; P.O. Box 1242, Addis Ababa, Ethiopia, E-mail
[email protected]; 2Department of Laboratory Technology, Faculty of Medicine, Jimma University, Jimma
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techniques and DNA isolation. Each sample was registered and numbered, and from this approximately one gram of faeces was persevered in 3 ml of sodium acetate-acetic-acid-formalin (SAF) for both direct and concentration method. Immediately after the stool was collected, one aliquot was examined by direct microscopy in normal saline and iodine and one aliquot that was preserved in SAF was examined by formol-ether concentration technique. Small portion of the fresh sample was mounted by saline and examined immediately for motile trophozoite stage and cysts. A drop of iodine was then added in order to see the nuclear structures as described previously (7). DNA Isolation One gram of faeces was suspended in 2 ml of 2 % pvpp (polyvinlypolypyrolidone) and was stored at –200C till DNA extraction. For isolation of DNA an aliquot of 200 µl of the suspension was separated in an eppendorf tube and was heated in heat block at 1000C for 10 minutes. After sodium dodecyl sulphate-proteinase K treatment with an equal volume (2 hours at 550C), DNA was isolated with QIAamp Tissue Kit spin columns (QIAGEN, Germany) (8). Amplification DNA amplification and colorimetric detection of the product was carried out as described previously (9-10). Briefly, two PCRs were performed on each DNA sample, one with E. histolytica- and the other with E. dispar – specific primers. One primer of each pair was labeled with digoxigenin (DIG). The primers for E. histolytica were P1 51 TCA AAA TGG TCG TCG TCT AGG C 31 and P2 51CAG TTA GAA ATT TTG ACT TGT A 31 while for E. dispar they were NP1 51 GGA TCC TCC AAA AAA TAA AGT TT 31 and NP2 51ACA GAA CGA TAT TGG ATA CCT AGT A 31. The PCR amplification condition was 2 minutes at 94ºC for denaturation; 35 cycles for 30 seconds at 94ºC, 30 seconds for 60ºC, and 30 seconds for 70ºC; final extension was for 2 minutes at 72ºC. The PCR product was visualized on 2 % agarose gel. Solution hybridization enzyme linked assay (SHELA) for detection of E. histolytica and E.dispar PCR products Colorimetric detection of the PCR product was achieved by hybridising with 5 pmol of 51- biotin- conjugated probes specific for E. histolytica and E. dispar, respectively. The specific probes sequences were p3bio 51GAG GTT CTT AGG AAA TCG AAA A31 (E. histolytica) and np3bio 51GGT GAG GTT GTA GCA GAG ATATT 31 (E. dispar). Denaturation (10 minutes at 99ºC) and hybridization (60 minutes at 60ºC) in the PCR machine was done after adding 1 µl biotinalated probe solution (5 pmol/µl) to 20 µl of PCR product. The ELISA was done on a NUNC Maxisorp titration plate, which was coated with streptavidin (1000 µg/ ml) as described before (10). The absorbance was read at 405 nm.
Result Out of the 228 stool specimens collected from outpatients coming to Jimma University Hospital, by microscopy 30 (13.2 %) were found to be infected with E. histolytica/E. dispar in which only 6 (2.6%) were trophozoites while 24 (10.5 %) were cysts. Upon confirmatory diagnostic test using polymerase chain reaction Solution Hybridization Enzyme Linked immunoassay (PCR-SHELA), 28 (12.3 %) turned out to be E. dispar out of the 30 microscopy positives (Table 1). No E. histolytica was detected. Two samples (6.7 %), which were positive by microscopy, were negative in the PCR. Examining the distribution of E. dispar with stool consistency revealed that significantly more E. dispar (P
