- Email: [email protected]
Comparison of two methods (microscopy and enzyme-linked immunosorbent assay) for the diagnosis of amebiasis
Experimental Parasitology 110 (2005) 322–326 www.elsevier.com/locate/yexpr
Comparison of two methods (microscopy and enzyme-linked immunosorbent assay) for the diagnosis of amebiasis Mehmet Tanyuksel a, Hasan Yilmaz b, Mustafa Ulukanligil c, Engin Araz a, Mutalip Cicek b, Ozgur Koru a, Zeynep Tas b, William A. Petri Jr. d,e,f,¤ a
Division of Medical Parasitology, Department of Microbiology and Clinical Microbiology, Gulhane Military Medical Academy, Etlik 06018, Ankara, Turkey b Department of Parasitology, Yuzuncu Yil University, Van, Turkey c Department of Microbiology, Harran University, Sanliurfa, Turkey d Department of Internal Medicine, University of Virginia, Charlottesville, VA 22908-1340, USA e Department of Pathology, University of Virginia, Charlottesville, VA 22908-1340, USA f Department of Microbiology, University of Virginia, Charlottesville, VA 22908-1340, USA Received 2 February 2005; received in revised form 17 February 2005; accepted 17 February 2005 Available online 23 March 2005
Abstract Diagnosis of amebiasis is usually performed on a clinical basis alone in most endemic countries having limited economic resources. This epidemiological study was conducted using modern diagnostic tests for amebiasis in the southeastern region of Turkey, an endemic area for amebiasis. The population of this study included patients with symptomatic diarrhea/dysentery attending both Yuzuncu Yil University, Van and Harran University, Sanliurfa, Turkey. A total of 380 stool specimens were collected and examined for Entamoeba by light microscopy (fresh, lugol, and trichrome staining) and stool antigen detection based- enzyme-linked immunosorbent assay (EIA) test (TechLab Entamoeba histolytica II). 24% (91/380) of stool specimens were positive for E. histolytica/ Entamoeba dispar trophozoites/cysts microscopically using trichrome staining. 13% (51/380) of the stool specimens were found to be positive for E. histolytica by the EIA test, including 15% (14/91) of microscopy (+) stool specimens and 13% (37/289) of microscopy (¡) stool specimens. Enteric parasites were common in these populations with 66% (251/380) of the study population harboring more than one parasite. In addition to the 13% (51/380) of patients determined to have E. histolytica by EIA, eighty-six patients (22.6%) had Blastocystis hominis, 54 (14.2%) Entamoeba coli, 44 (11.5%) Giardia lamblia, 16 (4.2%) Chilomastix mesnili, 15 (3.9%) Iodamoeba bütschlii, 12 (3.1%) Hymenolepis nana, 9 (2.3%) Endolimax nana, 9 (2.3%) Dientamoeba fragilis, and 8 (2.1%) had Ascaris lumbricoides. We concluded that E. histolytica infection was found in 13% of the patients presenting with diarrhea in Van and Sanliurfa Turkey. 2005 Elsevier Inc. All rights reserved. Keywords: Diagnosis; Entamoeba histolytica; Amebic colitis; Antigen detection; Diarrhea
1. Introduction Diarrhea and dysentery continue to be an important cause of disease with high morbidity and mortality rates, particularly in developing countries. Infection by Entamoeba histolytica is characterized by watery diarrhea *
Corresponding author. Fax: +1 434 924 0075. E-mail address: [email protected] (W.A. Petri Jr.).
0014-4894/$ - see front matter 2005 Elsevier Inc. All rights reserved. doi:10.1016/j.exppara.2005.02.012
that may progress to mucoid bloody diarrhea, also known as dysentery. However, some infections may be an asymptomatic. It has been estimated that each year over 50 million infections and 100,000 deaths amebiasis occur in the world (Anonymous, 1997). There are several methods used for amebiasis diagnosis and monitoring of E. histolytica infection. Microscopy is a relatively nonspeciWc but simple test for E. histolytica/Entamoeba dispar. Several newer molecular diagnostic tests have
M. Tanyuksel et al. / Experimental Parasitology 110 (2005) 322–326
become available that facilitate accurate diagnosis. The E. histolytica—speciWc antigen detection based-EIA test remains the principal modern means of diagnosis of infection in individuals. Our aim was to investigate the incidence of amebiasis using the antigen-detection test for diagnosis of E. histolytica infection in patients with diarrhea/dysentery in Van and Sanliurfa, Turkey in the eastern and southeastern region of Turkey.
323
dramatically in the last ten years due to rural to urban migration and there is a great need for improved infrastructure and residential areas. Geographically, it is located on very high rocky mountains. Hygienic conditions are poor. This area is endemic to many human parasites such as amebiasis, giardiasis, enterobiasis, and fascioliasis. 2.2. Ethical approval and informed consent
2. Materials and methods 2.1. Study site Sanliurfa province is in an underdeveloped region of southern Turkey. It is on the crossroads between the Mediterranean, the Anatolian plateau, and Mesopotamia (Fig. 1). The province is situated on a semi-arid plain at 550 m above sea level. The climate of the area has an average temperature of 18 °C and minimum’s of ¡12 °C in February and maximums of 47 °C in August. Average annual relative humidity is 49% and rainfall is 463 mm. The province is composed of 66 districts with a population of 813,575. Of the 66 districts, 43 (65%) are shantytowns, where 64% of the population live, and the remaining 23 districts (35%) are apartment areas, where 36% of the population live. Shantytowns are located in the city outskirts and usually consist of mudbrick houses around a small central courtyard. In these areas, sanitary conditions are poor, with household liquid and rubbish accumulating in the streets. On the other hand, a part of residents who have better socioeconomic conditions have settled in newly apartment buildings in the north of the city. Contrary to the shantytowns, apartment areas have good sanitation conditions. Van, Turkey’s eastern most province sharing the border with Iran, is northeast of Sirnak province and on the border of the Southeast Anatolian Project region. Van Lake the 5th largest lake in Europe, and one of the world’s highest lakes, is located there. Lake Van stands some 5500 ft (1650 m) above sea level and covers 3764 km2; it is fed by the streams of several rivers from the surrounding mountains. Van’s population has risen
Fig. 1. The map of Turkey, showing the study regions.
This study was reviewed and approved by the Human Investigation Committee of the University of Virginia and the IRB (local ethic committee). Permission to work in the community was obtained as individual written informed consent from subjects, parents, or guardians. Subjects enrolled in the study were from Van and Sanliurfa, Turkey. 2.3. Laboratory method A stool specimen from each subject was collected in plastic containers. Stool samples were examined by using a wet mount (fresh and lugol) and permanent stain (trichrome staining) microscopically for presence of Entamoeba in the laboratory. Antigen detection testing of stool specimens was performed by the E. histolytica II enzyme immunoassay (EIA) test (TechLab, Blacksburg, VA). This EIA uses the principal that the N-acetyl-D-galactoseamine-binding lectin (referred to as the galactose adhesin) is antigenically conserved but monoclonal antibodies can be used to distinguish the galactose adhesin from E. histolytica and E. dispar due to contain distinct epitopes. As a result, the test uses antibodies to the adhesin, and the conjugate is speciWc E. histolytica adhesin. At each run, two control wells (positive and negative controls) plus stool sample wells (as needed) were used for testing. All the samples were tested only one time. BrieXy, assay microtiter wells were incubated with one drop of monoclonal antibody (mAb)-enzyme conjugate and 200 L of diluted stool specimen for 2 h at room temperature. The contents of the well strips were then shaken out and washed Wve times. Residual liquid was then removed by striking the strip once against a paper towel, two drops of substrate were added to all test wells, and the strip was incubated at room temperature for 10 min. After the addition of one drop of stop solution, the optical density (OD) at 405 nm was measured with an automatic microplate spectrophotometer (Bio-Tek Instruments, USA) according to the description of the EIA kit included in the package insert. A positive result was deWned as an optical density reading >0.05 after subtraction of the negative control optical density. Statistical analysis was performed with StatsDirect (StatsDirect, Ver 2.20). To investigate the relations among the parameters, cross tables were produced.
324
M. Tanyuksel et al. / Experimental Parasitology 110 (2005) 322–326
DiVerences between two proportions were compared; p value was set at p 6 0.05.
3. Results As seen in Table 1, most of the study subjects from Van and Sanliurfa, Turkey suVered from diarrhea, malaise, fever, and loss of appetite. Microscopically, the results of examination (trichrome staining) of the subjects stool specimens were positive for E. histolytica/E. dispar trophozoites/cysts in 24% (91/380) of specimens. 13% (51/380) of specimens were found to be positive for E. histolytica antigen by the TechLab E. histolytica II EIA (Table 2). When results of the microscopy were compared to EIA, the antigen detection test revealed that 15% (14/91) of samples positive for E. histolytica/E. dispar contained E. histolytica by EIA. In conventional microscopic examination, E. histolytica/E. dispar trophozoites, cysts, or a mixture of cysts and trophozoites were observed in 4, 21, and 17% of all stool specimens, respectively. There was not a statistically signiWcant diVerence in the percentage of microscopy positive stool samples that were proven by TechLab EIA to be E. histolytica based on whether trophozoites or cysts were observed (p D 0.056). Erythrocytes were observed in 25% (13/51) of EIA positive stool samples (Table 3). We did not Wnd a signiWcant association between positivity and negativity of EIA in the presence of erythrocytes (p D 0.171). Trophozoites containing ingested erythrocytes were seen in only two samples. Also there was no statistically signiWcant diVerence between subjects with or without a positive E. histolytica EIA and a history of diarrhea (p D 0.450) or dysentery (p D 0.837). There were no signiWcant diVerences between age groups and positivity for E. histolytica (p D 0.286). There was however signiWcantly more dysentery in the
Table 2 Evaluation of microscopic Wndings (presence of E. histolytica/E. dispar trophozoite form) versus antigen detection (E. histolytica II EIA antigen detection kit) for detection of E. histolytica in stool samples (EIA) (n D 380) Microscopy
Entamoeba histolytica II EIA Positive n (%)
Negative n (%)
Total
Positive Negative
14 (28) 37 (73)
77 (23) 252 (77)
91 289
Total
51 (100)
329 (100)
380
Table 3 Findings of microscopic examination in 380 stool samples No. of patients with indicated result by Entamoeba histolytica II EIA Positive n (%)
Negative n (%)
Total
Presence of erythrocytes by the microscopic examination Positive 13 (25) 56 (17) Negative 38 (75) 273 (83)
69 311
Total
380
51 (100)
329 (100)
older than 30 age-group (p D 0.004) (Table 4). Twohundred Wfty-one subjects (66%) of the study population with intestinal parasites harbored more than one parasite (Table 5). Interestingly, Blastocystis hominis, Entamoeba coli, and Giardia lamblia were the most abundant intestinal protozoa in the stool by microscopic examination.
4. Discussion The degree to which infection with E. histolytica is a public health problem in the developing world is not certain, in part because of inaccuracies in classical methods of diagnosis. During the last decade there has been remarkable development in molecular-based diagnostic
Table 1 Most frequent clinical signs and symptoms reported by 380 patients admitted with diarrhoea/dysentery related to areas of origin of the study population in Van and Sanliurfa, Turkey Findings (signs and symptoms)
Van study site No. (%)
Sanliurfa study site No. (%)
Total No. (%)
Diarrhoea Fatigue Loss of appetite Fever Weight loss Dysentery Abdominal pain Malaise Urticeria Nause-vomiting Flatus Gastritis Psychomotor retardation Chest pain
255 (91.1) 127 (45.4) 83 (29.6) 46 (16.4) 65 (23.2) 51 (18.2) 45 (16.1) 10 (3.6) 7 (2.5) 6 (2.1) 1 (0.4) 1 (0.4) 1 (0.4) 1 (0.4)
87 (87.0) 60 (60.0) 56 (56.0) 66 (66.0) 46 (46.0) 10 (10.0) 6 (6.0) 7 (7.0) 1 (1.0) 1 (1.0) — — — —
342 (90.0) 187 (49.2) 139 (36.5) 112 (29.4) 111 (29.2) 61 (16.0) 51 (13.4) 17 (4.7) 8 (2.1) 7 (1.8) 1 (0.2) 1 (0.2) 1 (0.2) 1 (0.2)
M. Tanyuksel et al. / Experimental Parasitology 110 (2005) 322–326
325
Table 4 Distribution of the age-speciWc of the study population by EIA test, diarrhoea, and dysentery in Van and Sanliurfa, Turkey Age groups (years)
EIA
Diarrhoea
Positive n (%) 0–7 8–11 12–20 21–30 +31
17 (33) 6 (12) 3 (6) 8 (16) 17 (33)
Total
51
Negative n (%)
Positive n (%)
81 (25) 46 (14) 47 (14) 67 (20) 88 (27)
89 (26) 42 (12) 45 (13) 68 (20) 98 (29)
329
342
Table 5 Distribution of harbored intestinal parasites of the study population (n D 380) Parasite Blastocystis hominis Entamoeba coli Giardia lamblia Chilomastix mesnili Iodamoeba bütschlii Hymenolepis nana Endolimax nana Dientamoeba fragilis Ascaris lumbricoides Total
Van study site (#280) (%) 86 (31) 52 (18) 37 (13) 14 (5.0) 13 (4.6) 12 (4.3) 9 (3.2) 9 (3.2) 6 (2.1) 238
Sanliurfa study site (#100) (%) — 2 (2.0) 7 (7.0) 2 (2.0) — — — — 2 (2.0) 13
Total (#380) (%) 86 (23) 54 (14) 44 (12) 16 (4.2) 13 (3.4) 12 (3.1) 9 (2.3) 9 (2.3) 8 (2.1) 251
procedures to detect E. histolytica (Abd-Alla and Ravdin, 2002; Braga et al., 1998; Haque et al., 1998, 2000, 2003; Tanyuksel and Petri, 2003). Many studies have demonstrated the ability of E. histolytica EIA tests (Abd-Alla and Ravdin, 2002; Haque et al., 1998, 2000) to diVerentiate potentially invasive E. histolytica infection from commensal E. dispar infection. Diagnosis of E. histolytica had historically relied on microscopic examination of protozoan morphology. The microscopic diagnosis of amebiasis is neither speciWc nor sensitive. E. histolytica and E. dispar are indistinguishable morphologically (Tanyuksel and Petri, 2003). An additional limitation of microscopy is that fecal leukocytes often have been misidentiWed as E. histolytica cysts by laboratories (Doganci et al., 1997). In one study, the speciWcity of E. histolytica/E. dispar complex as determined by microscopy was only 9.5% in community laboratories when compared with the Entamoeba test and ProSpecT EIA antigen detection tests (Pillai et al., 1999). Conventional microscopy also has a disadvantage due to poor sensitivity that even in experienced hands may overlook 50% of infections, as we observed here (Table 2). The results of our work for the Wrst time provide an estimate of the contribution of amebiasis to disease among individuals presenting to Yuzuncu Yil University, Van and Harran University, Sanliurfa, Turkey. According to previous epidemiologic studies using
Dysentery Negative n (%)
Positive n (%)
9 (24) 10 (26) 5 (13) 7 (18) 7 (18)
10 (16) 3 (5) 8 (13) 14 (23) 26 (43)
38
61
Negative n (%) 88 (28) 49 (15) 42 (13) 61 (19) 79 (25) 319
only microscopy (native-lugol and staining with ironhematoxylen) prevalence rates of E. histolytica/E. dispar were 8–14% in Van (Yilmaz et al., 1997, 1999) and 15% in Sanliurfa (Ulukanligil et al., 2001). E. histolytica infection as determined by E. histolytica-speciWc EIA was present in 13% of patients with diarrhea and dysentery. Likely a reXection of the poor socioeconomic conditions under which the subjects lived, enteric parasites overall were common. Of the 380 individuals included in the study, 251 (66%) carried at least one parasite other than E. histolytica in their stool specimens (Table 5). Among them, B. hominis, E. coli, and G. lamblia were the most prevalent. Other identiWed intestinal parasites were Chilomastix mesnili, Iodamoeba bütschlii, Hymenolepis nana, Endolimax nana, Dientamoeba fragilis, and Ascaris lumbricoides. There were no signiWcant diVerences in infection rates for these parasites by sex, gender, and age (data not shown). While some have suggested that the presence of ingested red blood cells aids in the cytological diagnosis of amebiasis (Anaya-Velázquez and Barrios-Conejo, 2000), it was demonstrated that in some cases E. dispar is also noted to include red blood cells (Haque et al., 1995). In our study ingested red cells were a rare Wnding. Additionally the presence of erythrocytes in the stool is classically thought to be a reXection of invasive amebiasis. However, we found no signiWcant association between the presence of erythrocytes and E. histolytica in stool specimens. Other investigators have also identiWed E. histolytica infection in patients with non-bloody diarrhea (Haque et al., 2003). The EIA antigen detection test is more expensive than microscopic analysis but reduces inappropriate treatment and can be considered aVordable overall. The incidence and impact of amebiasis has been obscured by the prior inability to accurately diagnose amebiasis. Improved diagnostic tests are beginning to overturn some of the commonly held beliefs about amebiasis. As our experience with emerging E. histolytica infections increases and accurate laboratory-based diagnostic tests are employed, better management of this protozoan disease will become possible.
326
M. Tanyuksel et al. / Experimental Parasitology 110 (2005) 322–326
Acknowledgments The authors are grateful to Suzan Esin for lab assistance and Yavuz Sanisoglu and Cengiz Han Acikel for statistical analyses. This research was supported by NIH/FIRCA Grant TW-0060 17-01.
References Abd-Alla, M.D., Ravdin, J.I., 2002. Diagnosis of amoebic colitis by antigen capture ELISA in patients presenting with acute diarrhoea in Cairo, Egypt. Tropical Medicine and International Health 7, 365–370. Anaya-Velázquez, F., Barrios-Conejo, A., 2000. Cytological and immunological methods to identify Entamoeba histolytica and Entamoeba dispar trophozoites in patients from Guanajuato, Mexico. Archives of Medical Research 31, S32–S33. Anonymous. WHO/PAHO/UNESCO., 1997. Report of a consultation of experts on amebiasis. Weekly Epidemiology Record 72, 97–99. Braga, L.L., Mendonca, Y., Paiva, C.A., Sales, A., Cavalcante, A.L.M., Mann, B.J., 1998. Seropositivity for and intestinal colonization with Entamoeba histolytica and Entamoeba dispar in individuals in northeastern Brazil. Journal of Clinical Microbiology 36, 3044– 3045. Doganci, L., Tanyuksel, M., Gun, H., 1997. Overdiagnosis of intestinal amoebosis in Turkey. Lancet 350, 670. Haque, R., Mondal, D., Kirkpatrick, B.D., Akther, S., Farr, B.M., Sack, R.B., Petri Jr., W.A., 2003. Epidemiologic and clinical characteristics of acute diarrhea with emphasis on Entamoeba histolytica infec-
tions in preschool children in an urban slum of Dhaka, Bangladesh. American Journal of Tropical Medicine and Hygiene 69, 398–405. Haque, R., Neville, L.M., Hahn, P., Petri Jr., W.A., 1995. Rapid diagnosis of Entamoeba infection by using Entamoeba and Entamoeba histolytica stool antigen detection kits. Journal of Clinical Microbiology 33, 2558–2561. Haque, R., Mollah, N.U., Ali, I.K.M., Alam, K., Eubanks, A., Lyerly, D., Petri Jr., W.A., 2000. Diagnosis of amebic liver abscess and intestinal infection with the TechLab Entamoeba histolytica II antigen detection and antibody tests. Journal of Clinical Microbiology 38, 3235–3239. Haque, R., Ali, I.K.M., Akther, S., Petri Jr., W.A., 1998. Comparison of PCR, isoenzyme analysis, and antigen detection for diagnosis of Entamoeba histolytica infection. Journal of Clinical Microbiology 36, 449–452. Pillai, D.R., Keystone, J.S., Sheppard, D.C., MacLean, J.D., MacPherson, D.W., Kain, K.C., 1999. Entamoeba histolytica and Entamoeba dispar: epidemiology and comparison of diagnostic methods in a setting of nonendemicity. Clinical Infectious Disease 29, 315–1318. Tanyuksel, M., Petri jr., W.A., 2003. Laboratory diagnosis of amebiasis. Clinical Microbiology Reviews 16, 713–729. Ulukanligil, M., Bak•r, M., Aslan, G., Soran, M., Seyrek, A., 2001. Investigation of diarrhoeal agents among 0–5 years old children in Sanliurfa. Bulletin of Microbiology 35, 307–312 (in Turkish). Yilmaz, H., Akman, N., Godekmerdan, A., Goz, Y., 1997. EVect of socio-economic status and immigration on the distribution of intestinal parasites in 0–14 years old children. Van Medical Journal 4, 205–210 in Turkish. Yilmaz, H., Akman, N., Goz, Y., 1999. Distribution of intestinal parasites in two societies with diVerent socio-economic status in Van. Eastern Journal of Medicine 4, 16–19 (in Turkish).
Comparison of two methods (microscopy and enzyme-linked immunosorbent assay) for the diagnosis of amebiasis Mehmet Tanyuksel a, Hasan Yilmaz b, Mustafa Ulukanligil c, Engin Araz a, Mutalip Cicek b, Ozgur Koru a, Zeynep Tas b, William A. Petri Jr. d,e,f,¤ a
Division of Medical Parasitology, Department of Microbiology and Clinical Microbiology, Gulhane Military Medical Academy, Etlik 06018, Ankara, Turkey b Department of Parasitology, Yuzuncu Yil University, Van, Turkey c Department of Microbiology, Harran University, Sanliurfa, Turkey d Department of Internal Medicine, University of Virginia, Charlottesville, VA 22908-1340, USA e Department of Pathology, University of Virginia, Charlottesville, VA 22908-1340, USA f Department of Microbiology, University of Virginia, Charlottesville, VA 22908-1340, USA Received 2 February 2005; received in revised form 17 February 2005; accepted 17 February 2005 Available online 23 March 2005
Abstract Diagnosis of amebiasis is usually performed on a clinical basis alone in most endemic countries having limited economic resources. This epidemiological study was conducted using modern diagnostic tests for amebiasis in the southeastern region of Turkey, an endemic area for amebiasis. The population of this study included patients with symptomatic diarrhea/dysentery attending both Yuzuncu Yil University, Van and Harran University, Sanliurfa, Turkey. A total of 380 stool specimens were collected and examined for Entamoeba by light microscopy (fresh, lugol, and trichrome staining) and stool antigen detection based- enzyme-linked immunosorbent assay (EIA) test (TechLab Entamoeba histolytica II). 24% (91/380) of stool specimens were positive for E. histolytica/ Entamoeba dispar trophozoites/cysts microscopically using trichrome staining. 13% (51/380) of the stool specimens were found to be positive for E. histolytica by the EIA test, including 15% (14/91) of microscopy (+) stool specimens and 13% (37/289) of microscopy (¡) stool specimens. Enteric parasites were common in these populations with 66% (251/380) of the study population harboring more than one parasite. In addition to the 13% (51/380) of patients determined to have E. histolytica by EIA, eighty-six patients (22.6%) had Blastocystis hominis, 54 (14.2%) Entamoeba coli, 44 (11.5%) Giardia lamblia, 16 (4.2%) Chilomastix mesnili, 15 (3.9%) Iodamoeba bütschlii, 12 (3.1%) Hymenolepis nana, 9 (2.3%) Endolimax nana, 9 (2.3%) Dientamoeba fragilis, and 8 (2.1%) had Ascaris lumbricoides. We concluded that E. histolytica infection was found in 13% of the patients presenting with diarrhea in Van and Sanliurfa Turkey. 2005 Elsevier Inc. All rights reserved. Keywords: Diagnosis; Entamoeba histolytica; Amebic colitis; Antigen detection; Diarrhea
1. Introduction Diarrhea and dysentery continue to be an important cause of disease with high morbidity and mortality rates, particularly in developing countries. Infection by Entamoeba histolytica is characterized by watery diarrhea *
Corresponding author. Fax: +1 434 924 0075. E-mail address: [email protected] (W.A. Petri Jr.).
0014-4894/$ - see front matter 2005 Elsevier Inc. All rights reserved. doi:10.1016/j.exppara.2005.02.012
that may progress to mucoid bloody diarrhea, also known as dysentery. However, some infections may be an asymptomatic. It has been estimated that each year over 50 million infections and 100,000 deaths amebiasis occur in the world (Anonymous, 1997). There are several methods used for amebiasis diagnosis and monitoring of E. histolytica infection. Microscopy is a relatively nonspeciWc but simple test for E. histolytica/Entamoeba dispar. Several newer molecular diagnostic tests have
M. Tanyuksel et al. / Experimental Parasitology 110 (2005) 322–326
become available that facilitate accurate diagnosis. The E. histolytica—speciWc antigen detection based-EIA test remains the principal modern means of diagnosis of infection in individuals. Our aim was to investigate the incidence of amebiasis using the antigen-detection test for diagnosis of E. histolytica infection in patients with diarrhea/dysentery in Van and Sanliurfa, Turkey in the eastern and southeastern region of Turkey.
323
dramatically in the last ten years due to rural to urban migration and there is a great need for improved infrastructure and residential areas. Geographically, it is located on very high rocky mountains. Hygienic conditions are poor. This area is endemic to many human parasites such as amebiasis, giardiasis, enterobiasis, and fascioliasis. 2.2. Ethical approval and informed consent
2. Materials and methods 2.1. Study site Sanliurfa province is in an underdeveloped region of southern Turkey. It is on the crossroads between the Mediterranean, the Anatolian plateau, and Mesopotamia (Fig. 1). The province is situated on a semi-arid plain at 550 m above sea level. The climate of the area has an average temperature of 18 °C and minimum’s of ¡12 °C in February and maximums of 47 °C in August. Average annual relative humidity is 49% and rainfall is 463 mm. The province is composed of 66 districts with a population of 813,575. Of the 66 districts, 43 (65%) are shantytowns, where 64% of the population live, and the remaining 23 districts (35%) are apartment areas, where 36% of the population live. Shantytowns are located in the city outskirts and usually consist of mudbrick houses around a small central courtyard. In these areas, sanitary conditions are poor, with household liquid and rubbish accumulating in the streets. On the other hand, a part of residents who have better socioeconomic conditions have settled in newly apartment buildings in the north of the city. Contrary to the shantytowns, apartment areas have good sanitation conditions. Van, Turkey’s eastern most province sharing the border with Iran, is northeast of Sirnak province and on the border of the Southeast Anatolian Project region. Van Lake the 5th largest lake in Europe, and one of the world’s highest lakes, is located there. Lake Van stands some 5500 ft (1650 m) above sea level and covers 3764 km2; it is fed by the streams of several rivers from the surrounding mountains. Van’s population has risen
Fig. 1. The map of Turkey, showing the study regions.
This study was reviewed and approved by the Human Investigation Committee of the University of Virginia and the IRB (local ethic committee). Permission to work in the community was obtained as individual written informed consent from subjects, parents, or guardians. Subjects enrolled in the study were from Van and Sanliurfa, Turkey. 2.3. Laboratory method A stool specimen from each subject was collected in plastic containers. Stool samples were examined by using a wet mount (fresh and lugol) and permanent stain (trichrome staining) microscopically for presence of Entamoeba in the laboratory. Antigen detection testing of stool specimens was performed by the E. histolytica II enzyme immunoassay (EIA) test (TechLab, Blacksburg, VA). This EIA uses the principal that the N-acetyl-D-galactoseamine-binding lectin (referred to as the galactose adhesin) is antigenically conserved but monoclonal antibodies can be used to distinguish the galactose adhesin from E. histolytica and E. dispar due to contain distinct epitopes. As a result, the test uses antibodies to the adhesin, and the conjugate is speciWc E. histolytica adhesin. At each run, two control wells (positive and negative controls) plus stool sample wells (as needed) were used for testing. All the samples were tested only one time. BrieXy, assay microtiter wells were incubated with one drop of monoclonal antibody (mAb)-enzyme conjugate and 200 L of diluted stool specimen for 2 h at room temperature. The contents of the well strips were then shaken out and washed Wve times. Residual liquid was then removed by striking the strip once against a paper towel, two drops of substrate were added to all test wells, and the strip was incubated at room temperature for 10 min. After the addition of one drop of stop solution, the optical density (OD) at 405 nm was measured with an automatic microplate spectrophotometer (Bio-Tek Instruments, USA) according to the description of the EIA kit included in the package insert. A positive result was deWned as an optical density reading >0.05 after subtraction of the negative control optical density. Statistical analysis was performed with StatsDirect (StatsDirect, Ver 2.20). To investigate the relations among the parameters, cross tables were produced.
324
M. Tanyuksel et al. / Experimental Parasitology 110 (2005) 322–326
DiVerences between two proportions were compared; p value was set at p 6 0.05.
3. Results As seen in Table 1, most of the study subjects from Van and Sanliurfa, Turkey suVered from diarrhea, malaise, fever, and loss of appetite. Microscopically, the results of examination (trichrome staining) of the subjects stool specimens were positive for E. histolytica/E. dispar trophozoites/cysts in 24% (91/380) of specimens. 13% (51/380) of specimens were found to be positive for E. histolytica antigen by the TechLab E. histolytica II EIA (Table 2). When results of the microscopy were compared to EIA, the antigen detection test revealed that 15% (14/91) of samples positive for E. histolytica/E. dispar contained E. histolytica by EIA. In conventional microscopic examination, E. histolytica/E. dispar trophozoites, cysts, or a mixture of cysts and trophozoites were observed in 4, 21, and 17% of all stool specimens, respectively. There was not a statistically signiWcant diVerence in the percentage of microscopy positive stool samples that were proven by TechLab EIA to be E. histolytica based on whether trophozoites or cysts were observed (p D 0.056). Erythrocytes were observed in 25% (13/51) of EIA positive stool samples (Table 3). We did not Wnd a signiWcant association between positivity and negativity of EIA in the presence of erythrocytes (p D 0.171). Trophozoites containing ingested erythrocytes were seen in only two samples. Also there was no statistically signiWcant diVerence between subjects with or without a positive E. histolytica EIA and a history of diarrhea (p D 0.450) or dysentery (p D 0.837). There were no signiWcant diVerences between age groups and positivity for E. histolytica (p D 0.286). There was however signiWcantly more dysentery in the
Table 2 Evaluation of microscopic Wndings (presence of E. histolytica/E. dispar trophozoite form) versus antigen detection (E. histolytica II EIA antigen detection kit) for detection of E. histolytica in stool samples (EIA) (n D 380) Microscopy
Entamoeba histolytica II EIA Positive n (%)
Negative n (%)
Total
Positive Negative
14 (28) 37 (73)
77 (23) 252 (77)
91 289
Total
51 (100)
329 (100)
380
Table 3 Findings of microscopic examination in 380 stool samples No. of patients with indicated result by Entamoeba histolytica II EIA Positive n (%)
Negative n (%)
Total
Presence of erythrocytes by the microscopic examination Positive 13 (25) 56 (17) Negative 38 (75) 273 (83)
69 311
Total
380
51 (100)
329 (100)
older than 30 age-group (p D 0.004) (Table 4). Twohundred Wfty-one subjects (66%) of the study population with intestinal parasites harbored more than one parasite (Table 5). Interestingly, Blastocystis hominis, Entamoeba coli, and Giardia lamblia were the most abundant intestinal protozoa in the stool by microscopic examination.
4. Discussion The degree to which infection with E. histolytica is a public health problem in the developing world is not certain, in part because of inaccuracies in classical methods of diagnosis. During the last decade there has been remarkable development in molecular-based diagnostic
Table 1 Most frequent clinical signs and symptoms reported by 380 patients admitted with diarrhoea/dysentery related to areas of origin of the study population in Van and Sanliurfa, Turkey Findings (signs and symptoms)
Van study site No. (%)
Sanliurfa study site No. (%)
Total No. (%)
Diarrhoea Fatigue Loss of appetite Fever Weight loss Dysentery Abdominal pain Malaise Urticeria Nause-vomiting Flatus Gastritis Psychomotor retardation Chest pain
255 (91.1) 127 (45.4) 83 (29.6) 46 (16.4) 65 (23.2) 51 (18.2) 45 (16.1) 10 (3.6) 7 (2.5) 6 (2.1) 1 (0.4) 1 (0.4) 1 (0.4) 1 (0.4)
87 (87.0) 60 (60.0) 56 (56.0) 66 (66.0) 46 (46.0) 10 (10.0) 6 (6.0) 7 (7.0) 1 (1.0) 1 (1.0) — — — —
342 (90.0) 187 (49.2) 139 (36.5) 112 (29.4) 111 (29.2) 61 (16.0) 51 (13.4) 17 (4.7) 8 (2.1) 7 (1.8) 1 (0.2) 1 (0.2) 1 (0.2) 1 (0.2)
M. Tanyuksel et al. / Experimental Parasitology 110 (2005) 322–326
325
Table 4 Distribution of the age-speciWc of the study population by EIA test, diarrhoea, and dysentery in Van and Sanliurfa, Turkey Age groups (years)
EIA
Diarrhoea
Positive n (%) 0–7 8–11 12–20 21–30 +31
17 (33) 6 (12) 3 (6) 8 (16) 17 (33)
Total
51
Negative n (%)
Positive n (%)
81 (25) 46 (14) 47 (14) 67 (20) 88 (27)
89 (26) 42 (12) 45 (13) 68 (20) 98 (29)
329
342
Table 5 Distribution of harbored intestinal parasites of the study population (n D 380) Parasite Blastocystis hominis Entamoeba coli Giardia lamblia Chilomastix mesnili Iodamoeba bütschlii Hymenolepis nana Endolimax nana Dientamoeba fragilis Ascaris lumbricoides Total
Van study site (#280) (%) 86 (31) 52 (18) 37 (13) 14 (5.0) 13 (4.6) 12 (4.3) 9 (3.2) 9 (3.2) 6 (2.1) 238
Sanliurfa study site (#100) (%) — 2 (2.0) 7 (7.0) 2 (2.0) — — — — 2 (2.0) 13
Total (#380) (%) 86 (23) 54 (14) 44 (12) 16 (4.2) 13 (3.4) 12 (3.1) 9 (2.3) 9 (2.3) 8 (2.1) 251
procedures to detect E. histolytica (Abd-Alla and Ravdin, 2002; Braga et al., 1998; Haque et al., 1998, 2000, 2003; Tanyuksel and Petri, 2003). Many studies have demonstrated the ability of E. histolytica EIA tests (Abd-Alla and Ravdin, 2002; Haque et al., 1998, 2000) to diVerentiate potentially invasive E. histolytica infection from commensal E. dispar infection. Diagnosis of E. histolytica had historically relied on microscopic examination of protozoan morphology. The microscopic diagnosis of amebiasis is neither speciWc nor sensitive. E. histolytica and E. dispar are indistinguishable morphologically (Tanyuksel and Petri, 2003). An additional limitation of microscopy is that fecal leukocytes often have been misidentiWed as E. histolytica cysts by laboratories (Doganci et al., 1997). In one study, the speciWcity of E. histolytica/E. dispar complex as determined by microscopy was only 9.5% in community laboratories when compared with the Entamoeba test and ProSpecT EIA antigen detection tests (Pillai et al., 1999). Conventional microscopy also has a disadvantage due to poor sensitivity that even in experienced hands may overlook 50% of infections, as we observed here (Table 2). The results of our work for the Wrst time provide an estimate of the contribution of amebiasis to disease among individuals presenting to Yuzuncu Yil University, Van and Harran University, Sanliurfa, Turkey. According to previous epidemiologic studies using
Dysentery Negative n (%)
Positive n (%)
9 (24) 10 (26) 5 (13) 7 (18) 7 (18)
10 (16) 3 (5) 8 (13) 14 (23) 26 (43)
38
61
Negative n (%) 88 (28) 49 (15) 42 (13) 61 (19) 79 (25) 319
only microscopy (native-lugol and staining with ironhematoxylen) prevalence rates of E. histolytica/E. dispar were 8–14% in Van (Yilmaz et al., 1997, 1999) and 15% in Sanliurfa (Ulukanligil et al., 2001). E. histolytica infection as determined by E. histolytica-speciWc EIA was present in 13% of patients with diarrhea and dysentery. Likely a reXection of the poor socioeconomic conditions under which the subjects lived, enteric parasites overall were common. Of the 380 individuals included in the study, 251 (66%) carried at least one parasite other than E. histolytica in their stool specimens (Table 5). Among them, B. hominis, E. coli, and G. lamblia were the most prevalent. Other identiWed intestinal parasites were Chilomastix mesnili, Iodamoeba bütschlii, Hymenolepis nana, Endolimax nana, Dientamoeba fragilis, and Ascaris lumbricoides. There were no signiWcant diVerences in infection rates for these parasites by sex, gender, and age (data not shown). While some have suggested that the presence of ingested red blood cells aids in the cytological diagnosis of amebiasis (Anaya-Velázquez and Barrios-Conejo, 2000), it was demonstrated that in some cases E. dispar is also noted to include red blood cells (Haque et al., 1995). In our study ingested red cells were a rare Wnding. Additionally the presence of erythrocytes in the stool is classically thought to be a reXection of invasive amebiasis. However, we found no signiWcant association between the presence of erythrocytes and E. histolytica in stool specimens. Other investigators have also identiWed E. histolytica infection in patients with non-bloody diarrhea (Haque et al., 2003). The EIA antigen detection test is more expensive than microscopic analysis but reduces inappropriate treatment and can be considered aVordable overall. The incidence and impact of amebiasis has been obscured by the prior inability to accurately diagnose amebiasis. Improved diagnostic tests are beginning to overturn some of the commonly held beliefs about amebiasis. As our experience with emerging E. histolytica infections increases and accurate laboratory-based diagnostic tests are employed, better management of this protozoan disease will become possible.
326
M. Tanyuksel et al. / Experimental Parasitology 110 (2005) 322–326
Acknowledgments The authors are grateful to Suzan Esin for lab assistance and Yavuz Sanisoglu and Cengiz Han Acikel for statistical analyses. This research was supported by NIH/FIRCA Grant TW-0060 17-01.
References Abd-Alla, M.D., Ravdin, J.I., 2002. Diagnosis of amoebic colitis by antigen capture ELISA in patients presenting with acute diarrhoea in Cairo, Egypt. Tropical Medicine and International Health 7, 365–370. Anaya-Velázquez, F., Barrios-Conejo, A., 2000. Cytological and immunological methods to identify Entamoeba histolytica and Entamoeba dispar trophozoites in patients from Guanajuato, Mexico. Archives of Medical Research 31, S32–S33. Anonymous. WHO/PAHO/UNESCO., 1997. Report of a consultation of experts on amebiasis. Weekly Epidemiology Record 72, 97–99. Braga, L.L., Mendonca, Y., Paiva, C.A., Sales, A., Cavalcante, A.L.M., Mann, B.J., 1998. Seropositivity for and intestinal colonization with Entamoeba histolytica and Entamoeba dispar in individuals in northeastern Brazil. Journal of Clinical Microbiology 36, 3044– 3045. Doganci, L., Tanyuksel, M., Gun, H., 1997. Overdiagnosis of intestinal amoebosis in Turkey. Lancet 350, 670. Haque, R., Mondal, D., Kirkpatrick, B.D., Akther, S., Farr, B.M., Sack, R.B., Petri Jr., W.A., 2003. Epidemiologic and clinical characteristics of acute diarrhea with emphasis on Entamoeba histolytica infec-
tions in preschool children in an urban slum of Dhaka, Bangladesh. American Journal of Tropical Medicine and Hygiene 69, 398–405. Haque, R., Neville, L.M., Hahn, P., Petri Jr., W.A., 1995. Rapid diagnosis of Entamoeba infection by using Entamoeba and Entamoeba histolytica stool antigen detection kits. Journal of Clinical Microbiology 33, 2558–2561. Haque, R., Mollah, N.U., Ali, I.K.M., Alam, K., Eubanks, A., Lyerly, D., Petri Jr., W.A., 2000. Diagnosis of amebic liver abscess and intestinal infection with the TechLab Entamoeba histolytica II antigen detection and antibody tests. Journal of Clinical Microbiology 38, 3235–3239. Haque, R., Ali, I.K.M., Akther, S., Petri Jr., W.A., 1998. Comparison of PCR, isoenzyme analysis, and antigen detection for diagnosis of Entamoeba histolytica infection. Journal of Clinical Microbiology 36, 449–452. Pillai, D.R., Keystone, J.S., Sheppard, D.C., MacLean, J.D., MacPherson, D.W., Kain, K.C., 1999. Entamoeba histolytica and Entamoeba dispar: epidemiology and comparison of diagnostic methods in a setting of nonendemicity. Clinical Infectious Disease 29, 315–1318. Tanyuksel, M., Petri jr., W.A., 2003. Laboratory diagnosis of amebiasis. Clinical Microbiology Reviews 16, 713–729. Ulukanligil, M., Bak•r, M., Aslan, G., Soran, M., Seyrek, A., 2001. Investigation of diarrhoeal agents among 0–5 years old children in Sanliurfa. Bulletin of Microbiology 35, 307–312 (in Turkish). Yilmaz, H., Akman, N., Godekmerdan, A., Goz, Y., 1997. EVect of socio-economic status and immigration on the distribution of intestinal parasites in 0–14 years old children. Van Medical Journal 4, 205–210 in Turkish. Yilmaz, H., Akman, N., Goz, Y., 1999. Distribution of intestinal parasites in two societies with diVerent socio-economic status in Van. Eastern Journal of Medicine 4, 16–19 (in Turkish).