Objectives: Stool antigen assay has been shown to be as sensitive and specific as culture with isoenzyme analysis and to outperform microscopy for the detection of E.histolytica in endemic area. The aim of the present study is to investigate the presence of E.histolytica by direct microscopic examination and ELISA in stool samples, comparatively.Materials and methods: Between September 2010 and May 2011, a total of 975 stool samples of patients in different age groups were sent to microbiology laboratory of Kızıltepe General Hospital. Native-Lugol method and E.histolytica-specific antigen test (Adhesin Ag, Entamoeba CELISA Path) was applied to all stool samples.Results: E.histolytica/dispar cysts and/or trophozoites were observed in 21 out of 975 (2.2%) stool samples examined by native-Lugol method. In addition, E.histolytica-specific antigen in 975 stool specimens was investigated by ELISA. E.histolytica-specific antigen was determined in 4 patients which had E.histolytica/dispar cysts and/or trophozoites at direct microscopic examination. Although at direct microscopy of 3 patients E.histolytica/dispar cysts and/or trophozoites not observed, E.histolytica-specific antigen was found favorable. A total of 7 (0.7%) E.histolytica specific antigen was found in the patient’s stool samples. Patients with E.histolytica-specific antigen were treated.Conclusion: E.histolytica specific antigen in stool samples should be investigated to avoid unnecessary treatment

Alicem Tekin

Erkan Yula

Keramettin Yanık

Süleyman Durmaz

Türkan Toka Özer

Özcan Deveci

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T. T. Özer ve ark. Investigation of E.histolytica 294Dicle Tıp Derg / Dicle Med J Cilt / Vol 38, No 3, 294-297Dicle Tıp Dergisi /   2011; 38 (3): 294-297Dicle Medical Journal   doi: 10.5798/diclemedj.0921.2011.03.0034Yazışma Adresi /Correspondence: Türkan Toka ÖzerKızıltepe Devlet Hastanesi Mikrobiyoloji Laboratuarı, Kızıltepe, Mardin,Turkey     Email: tozer10@gmail.comCopyright © Dicle Tıp Dergisi 2011, Her hakkı saklıdır / All rights reservedORIGINAL ARTICLE / ÖZGÜN ARAŞTIRMAInvestigation of Entamoeba histolytica in stool specimens by direct microscopic examination and ELISA in a hospitalBir hastanede gaita örneklerinde direkt mikroskopik inceleme ve ELISA ile Entamoeba histolitika araştırılmasıTürkan Toka Özer 1, Erkan Yula 1, Özcan Deveci 2, Alicem Tekin 3, Süleyman Durmaz 4,Keramettin Yanık 51 Kızıltepe General Hospital, Department of Medical Microbiology, Mardin, Turkey2 Kızıltepe General Hospital, Department of Infectious Diseases, Mardin, Turkey3 Dicle University, Medical Faculty, Department of Medical Microbiology, Diyarbakır, Turkey4 Erciyes University, Medical Faculty, Department of Medical Microbiology, Kayseri, Turkey5 Amasya General Hospital, Department of Medical Microbiology, Amasya, TurkeyGeliş Tarihi / Received: 19.08.2011,  Kabul Tarihi / Accepted: 30.08.2011ÖZETAmaç: Dışkı örneklerinde antijen testinin, izoenzim ana-lizi ile birlikte kültür kadar duyarlı ve özgül bir test olduğu ve endemik bölgelerde mikroskopik bakıyı safdışı bıraktı-ğı gösterilmiştir. Bu çalışmanın amacı; dışkı örneklerinde direkt mikroskopik bakı ve ELISA yöntemi ile Entamoeba histolytica varlığının araştırılmasıdır.Gereç ve yöntem: Çalışmaya; Eylül 2010-Mayıs 2011 tarihleri arasında Kızıltepe Devlet Hastanesi Mikrobiyoloji laboratuvarına gönderilen, farklı yaş gruplarındaki hasta-lara ait 975 dışkı örneği dahil edildi. Tüm dışkı örnekleri-ne nativ-Lugol yöntemi ve E.histolytica-özgül antijen testi (Adhesin Ag, Entamoeba CELISA Path) uygulandı.Bulgular: Direkt bakıda nativ-Lugol yöntemi ile incelenen 975 dışkı örneğinin 21’inde (%2.2) Entamoeba histolytica/dispar kist ve/veya trofozoitleri görüldü. Ayrıca 975 dışkı örneğinde, ELISA yöntemi ile E.histolytica özgül antijeni araştırıldı. Direk mikroskopik bakıda Entamoeba histolyti-ca/dispar kist ve/veya trofozoitleri görülen hastaların sade-ce 4’ünde E.histolytica özgül antijeninin varlığı tespit edildi. Fakat 3 hastanın direk mikroskopik bakısında Entamoeba histolytica/dispar  kist  ve/veya  trofozoitleri  görülmezken E.histolytica özgül antijeni ise pozitif bulundu. Toplam ola-rak 7 (% 0.7) hastanın dışkı örneğinde E.histolytica özgül antijeninin varlığı tespit edildi. E.histolytica özgül antijeni saptanan olgulara uygun tedavi başlandı.Sonuç: Hastaların gereksiz tedavi almasını önlemek için dışkı  örneklerinde  E.histolytica  özgül  antijeninin  varlığı araştırılmalıdır.Anahtar kelimeler: Entamoeba histolytica/dispar, özgül antijen, ELISA, direk mikroskopik bakıABSTRACTObjectives:  Stool  antigen  assay  has  been  shown  to be as sensitive and specific as culture with isoenzyme analysis and to outperform microscopy for the detection of E.histolytica in endemic area. The aim of the present study is to investigate the presence of E.histolytica by di-rect microscopic examination and ELISA in stool samples, comparatively.Materials and methods: Between September 2010 and May 2011, a total of 975 stool samples of patients in dif-ferent age groups were sent to microbiology laboratory of Kızıltepe General Hospital. Native-Lugol method and E.histolytica-specific antigen test (Adhesin Ag, Entamoe-ba CELISA Path) was applied to all stool samples.Results:  E.histolytica/dispar  cysts  and/or  trophozoites were observed in 21 out of 975 (2.2%) stool samples ex-amined by native-Lugol method. In addition, E.histolytica-specific antigen in 975 stool specimens was investigated by ELISA. E.histolytica-specific antigen was determined in 4 patients which had E.histolytica/dispar cysts and/or trophozoites at direct microscopic examination. Although at  direct  microscopy  of  3  patients  E.histolytica/dispar cysts  and/or  trophozoites  not  observed,  E.histolytica-specific antigen was found favorable. A total of 7 (0.7%) E.histolytica specific antigen was found in the patient’s stool samples. Patients with E.histolytica-specific antigen were treated.Conclusion: E.histolytica specific antigen in stool sam-ples should be investigated to avoid unnecessary treat-ment.Key words: Entamoeba histolytica/dispar, specific anti-gen, ELISA, direct microscopic examination.T. T. Özer ve ark. Investigation of E.histolytica 295Dicle Tıp Derg / Dicle Med J Cilt / Vol 38, No 3, 294-297INTRODUCTIONEntamoeba  histolytica  is  the  causative  agent  of amoebiasis  and  is  globally  considered  a  leading parasitic  cause  of  human  mortality.  Clinical  fea-tures of amoebiasis due to E.histolytica range from asymptomatic  colonization  to  amebic  dysentery and  invasive  extraintestinal  amoebiasis,  which  is manifested  most  commonly  in  the  form  of  liver abscesses. Approximately 50 million people have invasive  disease,  resulting  in  100,000  deaths  per year.1 Although the parasite has a worldwide dis-tribution, high prevalence rates of more than 10% of the population have been reported from various developing countries.2 Entamoeba dispar appears to be about 10 times more common than E.histolytica, with most of the 500 million people infected with E.histolytica/E.dispar carrying E.dispar.1Entamoeba histolytica and Entamoeba dispar parasitize approximately 10% of the world popula-tion, of which 90% are asymptomatic infections.3 Infections of E.histolytica and E.dispar are often diagnosed by demonstrating cysts or trophozoites in a stool sample. A great number of methods for distinguishing  E.histolytica  from  E.dispar  have now been described in the literature.4 E.dispar and E.histolytica are morphologically indistinguishable from one another. Isoenzyme analysis is considered the “gold standard” for differentiating E.histolytica and E.dispar, but this method is not currently avail-able and not readily usable for routine diagnosis. More recently, several studies have been devoted to the development of new techniques either based on monoclonal antibodies or molecular biology meth-ods to successfully distinguish the two species in human feces.3 Stool antigen assay has been shown to be as sensitive and specific as culture with isoen-zyme analysis and to outperform microscopy for the detection of E.histolytica in areas of endemicity.5Reliable distinction would have a medical im-pact as until now, both infections are usually treated, whereas only approximately 10% (pathogenic infec-tions) need to be treated. This proportion drops too much lower levels in developed countries, where E.histolytica infection is not endemic and occurs mostly after travelling to areas of endemicity.3The present study was carried out to examine the prevalence and etiological agent of amoebia-sis in Kızıltepe. The main aim of this study was to demonstrate the importance of correctly identifying E.histolytica in order to avoid unnecessary treatment costs and delayed treatment of actual infection.MATERIALS AND METHODSCollection of stool samplesBetween September 2010 and May 2011, a total of 975 stool samples of patients of different age groups sent to Microbiology Laboratory of Kızıltepe Gen-eral Hospital were included in present study.Microscopic examinationStool  samples  were  investigated  by  native-Lugol examination. Lugol’s iodine was added to the stool smear and covered with a cover slip. Stool smears with saline or iodine examined microscopically at low (10X) and high (40X) magnifications within 15 minutes.The  identification  of  E.histolytica/dispar  tro-phozoites was made by the characteristic movement of the protozoan and the presence of phagocytized red blood cells. The identification of amebic cysts (E.histolytica/dispar)  was  based  on  morphologic characteristics, (10-15 μm, spherical form, mature tetranucleated cysts having a central endosome).Entamoeba antigen testFollowing  microscopic  examination  by  native-Lugol method, all of stool specimens were investi-gated for Entamoeba histolytica/dispar screening by Micro-ELISA method using commercial kits (Ad-hesin Ag, Entamoeba CELISA Path) regarding with the existence of adhesin antigens.RESULTSE.histolytica/dispar cysts and/or trophozoites were observed  in  21  out  of  975  (2.2%)  stool  samples examined  by  native-Lugol  method.  E.histolytica-specific antigen in 975 stool specimens was investi-gated by ELISA. E.histolytica-specific antigen was determined in 4 patients which had E.histolytica/dispar cysts and/or trophozoites at direct microscop-ic examination. Although at direct microscopy of 3 patients E.histolytica/dispar cysts and/or trophozo-ites not observed, E.histolytica-specific antigen was found favourable. A total of 7 (0.7%) E.histolytica specific  antigen  was  found  in  the  patient’s  stool T. T. Özer ve ark. Investigation of E.histolytica 296Dicle Tıp Derg / Dicle Med J Cilt / Vol 38, No 3, 294-297samples. Patients with E.histolytica-specific antigen had been treated.DISCUSSIONAmoebiasis is defined as infection with Entamoeba histolytica, regardless of associated symptomatolo-gy. In resource-rich nations, this parasitic protozoan is seen primarily in travelers to and emigrants from endemic areas. Infections range from asymptomatic colonization to amebic colitis and life-threatening abscesses. Importantly, disease may occur months to years after exposure. Although E.histolytica was previously thought to infect 10% of the world’s pop-ulation, 2 morphologically identical but genetically distinct and apparently non-pathogenic Entamoeba species are now recognized as causing most asymp-tomatic cases. To avoid unnecessary and possibly harmful therapies, clinicians should follow the diag-nostic and treatment guidelines of the World Health Organization.6Entameoba histolytica, 1 of the 2 Entamoeba species  with  similar  morphology  that  infect  hu-mans,  causes  invasive  intestinal  and  extraintesti-nal diseases, whereas Entamoeba dispar is found commensally and is non-invasive. Because of their morphologic similarity, E.histolytica and E.dispar cannot be differentiated microscopically. The anti-gens of E.histolytica and E.dispar, however, may be detected by the ELISA method. Previous studies have found that the detection of antigens in the stool samples is as sensitive and specific as cultures and isoenzyme analyses.7Studies  were  carried  out  at  a  mexican  pedi-atric  hospital  to  determine  the  ratio  between  the pathogenic  species  Entamoeba  histolytica  and non-pathogenic species E.dispar using an enzyme linked  immunosorbent  assay  (ELISA)  to  detect the lectin (1 galactose N-acetyl D-galactosamine) of E.histolytica in feces. A close correlation was noted  between  the  presence  of  the  E.histolytica lectin and clinical symptoms. In this study, amoe-bas were detected by microscopy in 120 children (either  E.histolytica  or  E.dispar).  But  while  al-most all (13/14) of the children with E.histolytica had clinical symptoms, dysentery-feces with mo-cus and blood, diarrhea, cramping abdominal pain, tenesmus rectal, flatulence, vomiting and headache, almost none (1/106) of the children infected with the non-pathogenic amoeba E.dispar had signs and symptoms. This suggests that much of the amoebia-sis diagnoses made in Mexico are, in fact, due to non-pathogenic E.dispar.8Malatyalı et al.9 reported that a total of 1449 stool samples were examined by native-Lugol and Trichrome staining, and 312 (22%) samples were positive  for  one  or  more  parasite  species. Addi-tionally,  22  (1.5%)  stool  samples  were  found  to be positive for the presence of E.histolytica/dispar cysts, and these samples were further examined by E.histolytica specific antigen based ELISA. As a re-sult, ELISA test gave negative reactions for all the samples. Also, there was no cross reaction with other luminal protozoa such as Escherichia coli, Giardia intestinalis,  Blastocystis  hominis  and  Iodamoeba butschlii. The data reveals that E.histolytica preva-lence may be lower than estimated.A  record  is  available  that  indicates  that E.histolytica  is  more  common  than  E.dispar  in Zonguldak. Mengeloglu et al.10 reported that ame-bic cysts were observed in 44 (0.37%) out of a total of 1720 stool specimens which were examined by direct microscopy. Entamoeba histolytica specific antigen was investigated with ELISA in the speci-mens that cysts were observed. Specific antigen was detected in 26 (59.1%) of these specimens. Because of the low sensitivity of direct microscopy in con-firming the prediagnosis of amoebiasis, it is neces-sary to perform ELISA on the specimens in order to determine whether the patient should be treated or to prevent patients from being given an unnecessary treatment.Zeyrek et al.11 reported that a total of 87 stool specimens  that  were  doubtful  using  the  native-Lugol method were examined by the E.histolytica specific sensu-lato antigen based ELISA test and the Trichrome staining method. Of these 87 stool specimens,  23  (26.4%)  specimens  were  positive for E.histolytica/E.dispar trophozoites/cysts micro-scopically using Trichrome staining and 19 (21.7%) of the stool specimens were found to be positive for the E.histolytica/E.dispar complex by the ELISA test.Tuncay et al.12 reported that stool samples of 9378 patients from different clinics, with several gastrointestinal  complaints  from  January  2004  to May 2006, were examined. All stool samples were examined  with  the  native-Lugol  method  and,  in suspicious cases, by Trichrome staining, cultivation T. T. Özer ve ark. Investigation of E.histolytica 297Dicle Tıp Derg / Dicle Med J Cilt / Vol 38, No 3, 294-297in Robinson’s medium and/or antigen detection in stool with the Entamoeba CELISA Path kit. Forty-one cases (0.44%), in which Entamoeba histolytica/Entamoeba dispar cysts and/or trophozoites were detected by at least one method, were found to be positive.In the world, the prevalence of E.histolytica is around 10% on average, but reaches up to 50% or 80% have been reported, in some regions. In Turkey the prevalence of E.histolytica is reported 0 to 17%. However, there are studies reporting high rates of detected between 43.2 to 77.7%.10In the light of earlier reports about the preva-lence of amoebiasis in such subjects, interpretation is very difficult because older data did not differenti-ate between morphologically identical species, one that is non-invasive (E.dispar) and are that is inva-sive (E.histolytica), but they have a high degree of divergence. It is very important to keep in mind that according to the older data, many E.histolytica in-fections were most probably confused with E.dispar due to limited data obtained from microscopic ex-aminations.13In conclusion, our findings are consistent with those previously reported studies in Turkey. Direct microscopic diagnosis of amoebiasis is not an effi-cient method for the diagnosis of E.histolytica, so we recommend stool antigen detection tests today offer a practical, sensitive, and specific method for the clinical laboratory to detect intestinal E.histolytica.REFERENCES1. Fotedar R, Stark D, Beebe N, Marriott D, Ellis J, Harkness J. Laboratory diagnostic techniques for Entamoeba species. Clin Microbiol Rev 2007;20(3):511-32.2. Stanley SL. Amoebiasis. Lancet 2003;361:1025-34.3. Gonin P, Trudel L. Detection and differentiation of Entam-oeba histolytica and Entamoeba dispar isolates in clinical samples by PCR and enzyme-linked immunosorbent assay. J Clin Microbiol 2003;41(1):237-41.4. Nesbitt RA, Mosha FW, Katki HA, Ashraf M, Assenga C, Lee  CM.  Amebiasis  and  comparison  of  microscopy  to ELISA technique in detection of Entamoeba histolytica and Entamoeba dispar. J Natl Med Assoc 2004;96(5):671-7.5.  Pillai  DR,  Keystone  JS,  Sheppard  DC,  MacLean  JD, MacPherson DW, Kain KC. Entamoeba histolytica and En-tamoeba dispar: epidemiology and comparison of diagnos-tic methods in a setting of nonendemicity. Clin Infect Dis 1999;29(5):1315-8.6. Pritt BS, Clark CG. Amebiasis. Mayo Clinic Proceedings 2008;83(10):1154-60.7. Delialioglu N, Aslan G, Ozturk C, Ozturhan H, Sen S, Eme-kdas G. Detection of Entamoeba histolytica antigen in stool samples in Mersin, Turkey. J Parasitol 2008;94(2):530-2.8. Redondo RB, Martinez Mendez LG, Baer G. Entamoeba histolytica and Entamoeba dispar: differentiation by En-zyme-Linked Immunosorbet Assay (ELISA) and its clini-cal  correlation  in  pediatrics  patients.  Parasitol  Latinoam 2006;61:37-42.9. Malatyalı E, Özçelik S, Çeliksöz A. The investigation of En-tamoeba histolytica Prevalance in some villages of Sivas by ELISA method. Turkiye Parazitol Derg 2011;35:6-9.10. Mengeloglu FZ, Aktas E, Kulah C, Comert Begendik F. De-tection of Entamoeba histolytica in stool specimens with the ELISA Method. Türkiye Parazitol Derg 2009;33(1):1-3.11. Yildiz Zeyrek F, Ozbilge H, Yüksel MF, Zeyrek CD, Sir-matel F. Parasitic fauna and the frequency of Entamoeba histolytica/Entamoeba dispar detected by ELISA in stool samples  in  Sanliurfa,  Turkey.  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Investigation of Entamoeba histolytica in stool specimens by direct microscopic examination and ELISA in a hospital

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