Entamoeba histolytica and Entamoeba dispar in the Northeast Brazil

MOTTA, C.M.S.; BELTRÃO, E.I.C.; AMORIM, R.V.S.; CARVALHO Jr., L.B.

doi:10.1590/S0036-46651997000600012

LETTER TO THE EDITOR

Entamoeba histolytica AND Entamoeba dispar IN THE NORTHEAST BRAZIL

Sir,

There was for many years a controversy concerning the relationship between the presence of protozoa Entamoeba histolytica and clinical symptoms. This species was easily identified by optical microscopy from Entamoeba coli in faecal samples. The latter species was definitely assumed not being pathogenic. However, among the strains of E. histolytica some were recognized as non-pathogenic. In 1970 decade isoenzymes electrophoretic patterns approaches were reported to distinguish these strains, namely, invasive and non-invasive strains of E. histolytica¹⁸. This concept brought back the proposal of Brumpt⁵ who described the non-pathogenic ameba as Entamoeba dispar. Clark & Diamond⁶ referred E. histolytica sensu lato as both species: the pathogenic species (E. histolytica sensu stricto) and the non-pathogenic species (E. dispar). Recently, molecular biology tools have been used to solve this polemic issue. Analysis of the gene encoding for a small subunit ribosomal RNA supported the existence of the two species¹¹. On the basis of this rRNA data, two primers were designed to produce polymerase chain reaction amplification from both E. histolytica and E. dispar. Primer specificity for the two ameba was assessed by theoretical and experimental data base of eukaryotic and prokaryotic DNAs. The amplified stretch encompasses a polimorphic Dde I restriction site which allowed, after cleavage of the fragment, E. histolytica and E. dispar to be distinguished. The reliability of this identification method was assessed comparing the results with those based on classic isoenzyme analysis. Furthermore, molecular biology of the hexokinase isoenzyme pattern was capable to distinguish pathogenic E. histolytica from non-pathogenic E. dispar¹⁴. In that study it was also observed that the four obtained bands in the isoenzyme patterns of these two forms of Entamoeba were correlated to four different cDNAs and that the four recombinant hexokinases produced in Escherichia coli comigrated with their natural counterparts. Molecular biology approaches have been recently used as an adjunct to microscopy, immunoassays and isoenzyme analysis in the diagnosis of amebic diseases^{3, 17}. However, immunoassays have been widely employed in the laboratorial routine. Gel diffusion precipitation test (GDP) was considered by some researchers^{10, 15, 21} to be one of the most reliable serological tests for diagnosis of amebiasis, since it is well correlated with the clinical course of symptomatic amebiasis. Enzyme-linked immunosorbent assay (ELISA) is also a tool for serodiagnostic method. Many antigens have been reported as specific for diagnosis of amebiasis such as E. histolytica trophozoite antigens, HM-1 IMSS^{13, 23}, pathogen-specific epitopes of the galactose adhesin of E. histolytica⁷, single recombinant E. histolytica antigen, P1-EIA⁸ and antigenic 170-Kda subunit of the amebal Gal/GalNAc-lectin¹⁹.

The controversial issue on amebic disease in the Northeast Brazil has been studied in the Laboratório de Imunopatologia Keizo Asami (LIKA) inside of a Brazilian and Japanese joint venture supported by the Japan International Cooperation Agency (JICA).

The prevalence of E. histolytica in developing countries is often assumed to be high, frequently without supporting data¹³. In Brazil, studies of E. histolytica among low income population have shown a difference among the North, Northeast and South regions. From 1988 to 1994, LIKA performed studies related to the prevalence of E. histolytica in Brazilian cities of North (Belém and Manaus), Northeast (Recife, Jaboatão, Bodocó, Palmares, Glória do Goitá and São Luiz) and South (São Paulo) regions. The used methodology varied since traditional serological test, such as GDP, and zymodemes to molecular biology, using genomic DNA amplification. The evaluated strains included both sex and age varying from newborn to adults. The results showed that the Amazon region (North) presented both E. histolytica and E. dispar with higher prevalence for E. histolytica while in the Northeast the E. dispar predominated^{1, 2, 12, 13, 20}.

Contradictory to these findings, Braga et al.⁴ studying the level of exposure of the Gonçalves Dias community in Fortaleza, Northeast of Brazil, to E. histolytica, reported the presence of serum antibodies specific for the Gal/GalNAc lectin. The results of this study demonstrated that this community in the Northeast Brazil is highly endemic for E. histolytica with infections rate similar to other developing nations.

In spite of Bragas results, different from those found in LIKA studies, the Northeast region seems to have a diverging parasitologic profile concerning the presence of E. histolytica and E. dispar. An attempt to identify cysts of E. histolytica and E. dispar in human stools, the application of recombinant DNA technology brings new horizons into the understanding of this protozoan parasite and its disease. The molecular biology techniques are fast, safe and outstanding approaches to overcame doubts and to answer questions about the occurrence of E. histolytica or E. dispar in the Northeast of Brazil. Polymerase chain reaction (PCR), random amplified polymorphic DNA (RAPD) and other methods have been used to identify and compare strains of Entamoeba^{9, 16, 22}. The Entamoeba characterization in the Northeast region of Brazil will be completely elucidated through the molecular biology techniques. For such reasons specific primers¹⁷ will be used, in studies at LIKA, to amplify the genetic material of both E. histolytica and E. dispar, searching for a complete identification of this protozoa in Northeast region of Brazil.

C.M.S. MOTTA. Departamento de Micologia,
CCB UFPE, Recife, PE, Brasil. E.I.C. BELTRÃO. Laboratório de Imunopatologia
Keizo Asami, UFPE, Recife, PE, Brasil. R.V.S. AMORIM. Laboratório de Imunopatologia
Keizo Asami, UFPE, Recife, PE, Brasil. L.B. CARVALHO Jr. Laboratório de Imunopatologia
Keizo Asami, UFPE, Recife, PE, Brasil.

Correspondence to: Prof. Dr. Luiz Bezerra de Carvalho Jr., Laboratório de Imunopatologia Keizo Asami (LIKA). Av. Prof. Moraes Rêgo s/n, Cidade Universitária, Recife, 50670-901, PE, Brasil.

Recebido para publicação em 07/11/1997

Aceito para publicação em 25/11/1997

1. ACA, I. S.; FRANÇA Jr., E.; NOZAKI, T. et al. Entamoeba histolytica zymodemes in children of Osasco, Săo Paulo. Rev. Inst. Med. trop. S. Paulo, 35: 581-582, 1993.
2. ACA, I. S.; KOBAYASHI, S.; CARVALHO Jr., L. B. et al. Prevalence and pathogenicity of Entamoeba histolytica in three different regions of Pernambuco, Northeast Brazil. Rev. Inst. Med. trop. S. Paulo, 36: 519-524, 1994.
3. ACUNA-SOTO, R.; SAMUELSON, J.; DE GIROLANI, P. et al. Application of the polymerase chain reaction to the epidemiology of pathogenic and nonpathogenic Entamoeba histolytica Amer. J. trop. Med. Hyg., 48: 58-70, 1993.
4. BRAGA, L. L.; LIMA. A. A. M.; SEARS, C. L. et al. Seroepidemiology of Entamoeba histolytica in a slum in Northeast Brazil. Amer. J. trop. Med. Hyg., 55: 693-697, 1996.
5. BRUMPT, E. Étude sommarie de lEntamoeba dispar n.sp. amibe ŕ kystes quadrinuclées, parasite de l'homme. Bull. Acad. Méd. (Paris), 94: 943-952, 1925.
6. CLARK, C. G. & DIAMOND, L. S. Entamoeba histolytica: a explanation for the reported conversion of nonpathogenic amoeba to the pathogenic form. Exp. Parasit., 77: 456-460, 1993.
7. HAQUE, R.; KRESS, K.; WOOD, S. et al. Diagnosis of pathogenic Entamoeba histolytica infection using a stool ELISA based on monoclonal antibodies to the galactose-specific adhesin. J. infect. Dis., 167: 247-249, 1993.
8. LOTTER, H.; JACKSON, T. F & TANNICH, E. Evaluation of three serological tests for the detection of antiamebic antibodies applied to sera of patients from an area endemic for amebiasis. Trop. Med. Parasit., 46: 180-183, 1995.
9. MACKENSEDT, U. & JOHNSON, A. M. Genetic differentiation of pathogenic and non-pathogenic strains of Entamoeba histolytica by random amplified polymorphic DNA polymerase chain reaction. Parasit. Res., 81: 217-221, 1995.
10. MADDISON, S. E. Characterization of Entamoeba histolytica antigen-antibody reaction by gel diffusion. Exp. Parasit., 16: 224-235, 1965.
11. NOVATI, S.; SIRONI, M.; GRANATA, S. et al. Direct sequencing of the PCR amplified SSU rRNA gene of Entamoeba dispar and the design of primers for rapid differentiation from Entamoeba histolytica Parasitology, 112: 363-369, 1996.
12. NOZAKI, T.; ACA, I. S.; OKUZAWA, E. et al. Zymodemes of Entamoeba histolytica isolated in the Amazon and the north-east of Brazil. Trans. roy. Soc. trop. Med. Hyg., 84: 387-388, 1990.
13. OKASAKI, M.; MIRANDA, P.; NETO, J. et al. Parasitological and serological studies on amoebiasis and other intestinal parasitic infections in Recife and its suburban area, Northeast Brazil. Rev. Inst. Med. trop. S. Paulo, 30: 313-321, 1988.
14. ORTNER, S.; CLARK, C. G.; BINDER, M. et al. Molecular biology of the hexokinase isoenzyme pattern that distinguishes pathogenic Entamoeba histolytica from nonpathogenic Entamoeba dispar Molec. Biochem. Parasit., 86: 85-94, 1997.
15. PATTERSON, M.; HEALY, G. R. & SHABOT, J. M. Serologic testing for amoebiasis. Gastroenterology, 78: 136-141, 1980.
16. RIVERA, W. L.; TACHIBANA, H.; SILVA-TAHAT, M. R. et al. Differentiation of Entamoeba histolytica and E. dispar DNA from cysts present in stool specimens by polymerase chain reaction: its field application in the Philippines. Parasit. Res., 82: 585-589, 1996.
17. SANUKI, J.; ASAI, T.; OKUZAWA, E. et al. Identification of Entamoeba histolytica and E. dispar cysts in stool by polymerase chain reaction. Parasit. Res., 83: 96-98, 1997.
18. SARGEAUNT, P. G. & WILLIAMS, J. E. Electrophoretic isoenzyme patterns of Entamoeba histolytica and Entamoeba coli Trans. roy. Soc. trop. Med. Hyg., 72: 164-166, 1978.
19. SHENAI, B. R.; KOMALAM, B. L.; ARVIND, A. S. et al. Recombinant antigen-based avidin-biotin microtiter enzyme-linked immunosorbent assay for serodiagnosis of invasive amebiasis. J. clin. Microbiol., 34: 828-833, 1996.
20. TACHIBANA, H.; KOBAYASHI, S.; PAZ, K. C. et al. Analysis of pathogenicity by restriction-endonuclease digestion of amplified genomic DNA of Entamoeba histolytica isolated in Pernambuco, Brazil. Parasit. Res., 78: 433-436, 1992.
21. TAKEUCHI, T.; KOBAYASHI, S. & TATENO, S. Evaluation of serologic testings of amoebiasis. Jap. J. Parasit., 34: 75, 1985.
22. TROLL, H.; MARTI, H. & WEISS, N. Simple differential detection of Entamoeba histolytica and Entamoeba dispar in fresh stool specimens by sodium acetate-acetic acid-formalin concentration and PCR. J. clin. Microbiol., 35: 1701-1705, 1997.
23. WONSIT, R.; THAMMAPALERD, N.; THARAVANIJ, S. et al. Enzyme-linked immunosorbent assay based on monoclonal and polyclonal antibodies for the detection of Entamoeba histolytica antigens in faecal specimens. Trans. roy. Soc. trop. Med. Hyg., 86: 166-169, 1992.

Publication Dates

Publication in this collection
09 Oct 1998
Date of issue
Nov 1997

History

Received
07 Nov 1997
Accepted
25 Nov 1997

This work is licensed under a Creative Commons Attribution-NonCommercial 4.0 International License.

[1] 1. ACA, I. S.; FRANÇA Jr., E.; NOZAKI, T. et al. Entamoeba histolytica zymodemes in children of Osasco, Săo Paulo. Rev. Inst. Med. trop. S. Paulo, 35: 581-582, 1993.

[2] 2. ACA, I. S.; KOBAYASHI, S.; CARVALHO Jr., L. B. et al. Prevalence and pathogenicity of Entamoeba histolytica in three different regions of Pernambuco, Northeast Brazil. Rev. Inst. Med. trop. S. Paulo, 36: 519-524, 1994.

[3] 3. ACUNA-SOTO, R.; SAMUELSON, J.; DE GIROLANI, P. et al. Application of the polymerase chain reaction to the epidemiology of pathogenic and nonpathogenic Entamoeba histolytica Amer. J. trop. Med. Hyg., 48: 58-70, 1993.

[4] 4. BRAGA, L. L.; LIMA. A. A. M.; SEARS, C. L. et al. Seroepidemiology of Entamoeba histolytica in a slum in Northeast Brazil. Amer. J. trop. Med. Hyg., 55: 693-697, 1996.

[5] 5. BRUMPT, E. Étude sommarie de lEntamoeba dispar n.sp. amibe ŕ kystes quadrinuclées, parasite de l'homme. Bull. Acad. Méd. (Paris), 94: 943-952, 1925.

[6] 6. CLARK, C. G. & DIAMOND, L. S. Entamoeba histolytica: a explanation for the reported conversion of nonpathogenic amoeba to the pathogenic form. Exp. Parasit., 77: 456-460, 1993.

[7] 7. HAQUE, R.; KRESS, K.; WOOD, S. et al. Diagnosis of pathogenic Entamoeba histolytica infection using a stool ELISA based on monoclonal antibodies to the galactose-specific adhesin. J. infect. Dis., 167: 247-249, 1993.

[8] 8. LOTTER, H.; JACKSON, T. F & TANNICH, E. Evaluation of three serological tests for the detection of antiamebic antibodies applied to sera of patients from an area endemic for amebiasis. Trop. Med. Parasit., 46: 180-183, 1995.

[9] 9. MACKENSEDT, U. & JOHNSON, A. M. Genetic differentiation of pathogenic and non-pathogenic strains of Entamoeba histolytica by random amplified polymorphic DNA polymerase chain reaction. Parasit. Res., 81: 217-221, 1995.

[10] 10. MADDISON, S. E. Characterization of Entamoeba histolytica antigen-antibody reaction by gel diffusion. Exp. Parasit., 16: 224-235, 1965.

[11] 11. NOVATI, S.; SIRONI, M.; GRANATA, S. et al. Direct sequencing of the PCR amplified SSU rRNA gene of Entamoeba dispar and the design of primers for rapid differentiation from Entamoeba histolytica Parasitology, 112: 363-369, 1996.

[12] 12. NOZAKI, T.; ACA, I. S.; OKUZAWA, E. et al. Zymodemes of Entamoeba histolytica isolated in the Amazon and the north-east of Brazil. Trans. roy. Soc. trop. Med. Hyg., 84: 387-388, 1990.

[13] 13. OKASAKI, M.; MIRANDA, P.; NETO, J. et al. Parasitological and serological studies on amoebiasis and other intestinal parasitic infections in Recife and its suburban area, Northeast Brazil. Rev. Inst. Med. trop. S. Paulo, 30: 313-321, 1988.

[14] 14. ORTNER, S.; CLARK, C. G.; BINDER, M. et al. Molecular biology of the hexokinase isoenzyme pattern that distinguishes pathogenic Entamoeba histolytica from nonpathogenic Entamoeba dispar Molec. Biochem. Parasit., 86: 85-94, 1997.

[15] 15. PATTERSON, M.; HEALY, G. R. & SHABOT, J. M. Serologic testing for amoebiasis. Gastroenterology, 78: 136-141, 1980.

[16] 16. RIVERA, W. L.; TACHIBANA, H.; SILVA-TAHAT, M. R. et al. Differentiation of Entamoeba histolytica and E. dispar DNA from cysts present in stool specimens by polymerase chain reaction: its field application in the Philippines. Parasit. Res., 82: 585-589, 1996.

[17] 17. SANUKI, J.; ASAI, T.; OKUZAWA, E. et al. Identification of Entamoeba histolytica and E. dispar cysts in stool by polymerase chain reaction. Parasit. Res., 83: 96-98, 1997.

[18] 18. SARGEAUNT, P. G. & WILLIAMS, J. E. Electrophoretic isoenzyme patterns of Entamoeba histolytica and Entamoeba coli Trans. roy. Soc. trop. Med. Hyg., 72: 164-166, 1978.

[19] 19. SHENAI, B. R.; KOMALAM, B. L.; ARVIND, A. S. et al. Recombinant antigen-based avidin-biotin microtiter enzyme-linked immunosorbent assay for serodiagnosis of invasive amebiasis. J. clin. Microbiol., 34: 828-833, 1996.

[20] 20. TACHIBANA, H.; KOBAYASHI, S.; PAZ, K. C. et al. Analysis of pathogenicity by restriction-endonuclease digestion of amplified genomic DNA of Entamoeba histolytica isolated in Pernambuco, Brazil. Parasit. Res., 78: 433-436, 1992.

[21] 21. TAKEUCHI, T.; KOBAYASHI, S. & TATENO, S. Evaluation of serologic testings of amoebiasis. Jap. J. Parasit., 34: 75, 1985.

[22] 22. TROLL, H.; MARTI, H. & WEISS, N. Simple differential detection of Entamoeba histolytica and Entamoeba dispar in fresh stool specimens by sodium acetate-acetic acid-formalin concentration and PCR. J. clin. Microbiol., 35: 1701-1705, 1997.

[23] 23. WONSIT, R.; THAMMAPALERD, N.; THARAVANIJ, S. et al. Enzyme-linked immunosorbent assay based on monoclonal and polyclonal antibodies for the detection of Entamoeba histolytica antigens in faecal specimens. Trans. roy. Soc. trop. Med. Hyg., 86: 166-169, 1992.