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    Typical polymerase chain reaction amplifications of DNA from trophozoites harvested from 10 stool samples (2–6 and 9–13) and using primers for Entamoeba histolytica (p11/p12) and E. dispar (p13/p14). Lanes a and b = amplifications for the primers p13/p14 (≃ 100 basepairs) and p11/p12, respectively. Lanes 1 and 8 = positive controls for E. dispar; lanes 7 and 14 = positive controls for E. histolytica; lanes M = molecular mass marker.

  • 1

    Walsh JA, 1986. Problems in recognition and diagnosis of amoebiasis: estimation of the global magnitude of morbidity and mortality. Rev Infect Dis 8 :228–238.

    • Search Google Scholar
    • Export Citation
  • 2

    World Health Organization, 1997. Amoebiasis. Report on the WHO/Pan American Health Organization/ UNESCO Expert Consultation, Mexico City. Wkly Epidemiol Rec 72 :97–100.

    • Search Google Scholar
    • Export Citation
  • 3

    Okazaki M, Miranda P, Neto J, Diegues V, Alves J, Cauas M, Tanabe M, Kobayashi S, Tateno S, Takeuchi T, 1988. Parasitological and serological studies on amoebiasis and other intestinal parasitic infections in Recife and its suburban area, northeast Brazil. Rev Inst Med Trop Sao Paulo 30 :313–321.

    • Search Google Scholar
    • Export Citation
  • 4

    Nozaki T, Aca IS, Okuzawa E, Magalhães M, Tateno S, Takeuchi T, 1990. Zymodemes of Entamoeba histolytica isolated in the Amazon and the northeast of Brazil. Trans R Soc Trop Med Hyg 84 :387–388.

    • Search Google Scholar
    • Export Citation
  • 5

    Aca IS, França JR, Nozaki T, Freitas GB, Tateno S, 1993. Entamoeba histolytica zymodemes in children of Osasco. Rev Inst Med Trop Sao Paulo 35 :581–582.

    • Search Google Scholar
    • Export Citation
  • 6

    Aca IS, Kobayashi S, Carvalho LB Jr, Tateno S, Takeuchi T, 1994. Prevalence and pathogenicity of Entamoeba histolytica in three different regions of Pernambuco. Rev Inst Med Trop Sao Paulo 36 :519–524.

    • Search Google Scholar
    • Export Citation
  • 7

    Tachibana H, Kobayashi S, Paz KC, Aca IS, Tateno S, Ihara S, 1992b. Analysis of pathogenicity by restriction-endonuclease digestion of amplified genomic DNA of Entamoeba histolytica isolated in Pernambuco, Brazil. Parasitol Res 78 :433–436.

    • Search Google Scholar
    • Export Citation
  • 8

    Braga LL, Lima AL, Sears CL, Newman RD, Wuhib T, Paiva CA, Guerrant RL, Mann BJ, 1996. Seroepidemiology of Entamoeba histolytica in a slum in northeast Brazil. Rev Inst Med Trop Sao Paulo 55 :693–697.

    • Search Google Scholar
    • Export Citation
  • 9

    Maddison SE, 1965. Characterization of Entamoeba histolytica antigen-antibody reaction by gel diffusion. Exp Parasitol 16 :224–235.

  • 10

    Patterson M, Healy GR, Shabot JM, 1980. Serologic testing for amoebiasis. Gastroenterology 78 :136–141.

  • 11

    Singh B, 1997. Molecular methods for diagnosis and epidemiological studies of parasitic infections. Int J Parasitol 27 :1135–1145.

  • 12

    Wonsit R, Thammapalerd N, Tharavanij S, Radomyos P, Bunnag D, 1992. Enzyme-linked immunosorbent assay based on monoclonal and polyclonal antibodies for the detection of Entamoeba histolytica antigens in faecal specimens. Trans R Soc Trop Med Hyg 86 :166–169.

    • Search Google Scholar
    • Export Citation
  • 13

    Mackenstedt U, Johnson AM, 1995. Genetic differentiation of pathogenic and non-pathogenic strains of Entamoeba histolytica by random amplified polymorphic DNA-polymerase chain reaction. Parasitol Res 81 :217–221.

    • Search Google Scholar
    • Export Citation
  • 14

    Haque R, Kress K, Wood S, Jackson TFHG, Lyerly D, Petri WA Jr, 1993. 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.

    • Search Google Scholar
    • Export Citation
  • 15

    Shenai BR, Komalam BL, Arvind AS, 1996. Recombinant antigen-based avidin-biotin microtiter enzyme-linked immunosorbent assay for serodiagnosis of invasive amebiasis. J Clin Microbiol 34 :828–833.

    • Search Google Scholar
    • Export Citation
  • 16

    Mirelman D, Nuchamowitz Y, Stolarsky T, 1997. Comparison of enzyme-linked immunosorbent assay-based kits and PCR amplification of rRNA genes for simultaneous detection of Entamoeba histolytica and E. dispar. J Clin Microbiol 5 :2405–2407.

    • Search Google Scholar
    • Export Citation
  • 17

    Clark CG, Diamond LS, 1991. Ribosomal RNA genes of ‘pathogenic’ and ‘nonpathogenic’ Entamoeba histolytica are distinct. Mol Biochem Parasitol 47 :297–302.

    • Search Google Scholar
    • Export Citation
  • 18

    Novati S, Sironi M, Granata S, Bruno A, Gatti S, Scaglia M, Bandi C, 1996. 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.

    • Search Google Scholar
    • Export Citation
  • 19

    Myjak P, Kur J, Pietkiewicz H, 1997. Usefulness of new DNA extraction procedure for PCR technique in species identification of Entamoeba isolates. Wiad Parazytol 43 :163–170.

    • Search Google Scholar
    • Export Citation
  • 20

    Tachibana H, Kobayashi S, Nagakura K, Kaneda Y, Takeuchi T, 1991. Reactivity of monoclonal antibodies to species-specific antigens of Entamoeba histolytica. J Protozool 38 :329–334.

    • Search Google Scholar
    • Export Citation
  • 21

    Cheng XJ, Tachibana H, Kobayashi S, Kaneda Y, Huang MY, 1993. Pathogenicity of Entamoeba histolytica isolates from Shangai, China. Parasitol Res 79 :608–610.

    • Search Google Scholar
    • Export Citation
  • 22

    Rivera WL, Tachibana H, Silva-Tahat MRA, Haruki U, Kanbara H, 1996. Differentiation of Entamoeba histolytica and E. dispar DNA from cysts present in stool specimens by polymerase chain reaction: its field application in the Philippines. Parasitol Res 82 :585–589.

    • Search Google Scholar
    • Export Citation
  • 23

    Tachibana H, Cheng XJ, Kobayashi S, Matsubayashi N, Goth S, Matsubayashi K, 2001. High prevalence of infection with Entamoeba dispar but not E. histolytica, in captive macaques. Parasitol Res 87 :14–17.

    • Search Google Scholar
    • Export Citation
  • 24

    Tachibana H, Kobayashi S, Takekoshi M, Ihara S, 1991. Distinguishing pathogenic and nonpathogenic isolates of Entamoeba histolytica by polymerase chain reaction. J Infect Dis 164 :825–826.

    • Search Google Scholar
    • Export Citation
  • 25

    Zaman S, Khoo J, Ng SW, Ahmed R, Khan MA, Hussain R, Zaman V, 2000. Direct amplification of Entamoeba histolytica DNA from amoebic liver abscess pus using polymerase chain reaction. Parasitol Res 86 :101–109.

    • Search Google Scholar
    • Export Citation
  • 26

    Hoffman WA, Pons JA, Janer JL, 1934. The sedimentation concentration method in schistosomiasis mansoni. J Public Health Trop Med 9 :283–298.

    • Search Google Scholar
    • Export Citation
  • 27

    Ritchie LS, 1948. An ether sedimentation technique for routine stool examination. Bull USA Med Dept 8 :326.

  • 28

    Robinson GI, 1968. The laboratory diagnosis of human parasitic amoeba. Trans R Soc Trop Med Hyg 62 :285–293.

  • 29

    Haque R, Neville LM, Hahn P, Petri WA Jr, 1995. Rapid diagnosis of Entamoeba infection by using Entamoeba and Entamoeba histolytica stool antigen detection kits. J Clin Microbiol 33 :2558–2561.

    • Search Google Scholar
    • Export Citation
  • 30

    Sehgal R, Abd-Alla M, Moody AH, Chiodini PL, Ackers JP, 1995. Comparison of two media for the isolation and short-term culture of Entamoeba histolytica and E. dispar. Trans R Soc Trop Med Hyg 89 :394.

    • Search Google Scholar
    • Export Citation
  • 31

    Alencar LCA, Magalhães V, Melo VM, Aca IS, Magalhães M, Kobayashi S, 1996. Ausência de amebíase invasiva em aidéticos homossexuais masculinos. Rev Soc Bras Med Trop 29 :319–322.

    • Search Google Scholar
    • Export Citation
  • 32

    Lima TA, Aca IS, Magalhães V, Melo V, Lima RA, Magalhães M, 1998. Etiologia dos abscessos hepáticos criptogenéticos. Arq Bras Med 72 :141–145.

    • Search Google Scholar
    • Export Citation

 

 

 

DETERMINATION OF THE PREVALENCE OF ENTAMOEBA HISTOLYTICA AND E. DISPAR IN THE PERNAMBUCO STATE OF NORTHEASTERN BRAZIL BY A POLYMERASE CHAIN REACTION

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  • 1 Laboratório de Imunopatologia Keizo Asami; Departamento de Medicina Tropical; Departamento de Medicina Social; Departamento de Genética, e Departamento de Bioquímica, Universidade Federal de Pernambuco, Recife, Pernambuco, Brazil; Departamento de Pesca, Universidade Federal Rural de Pernambuco, Recife, Brazil

Previous studies using methods varying from traditional serologic tests to molecular biology techniques have shown that in northeastern Brazil, Entamoeba dispar was more prevalent than E. histolytica. In this study, the prevalence was established by using E. histolytica stool antigen detection kits and a polymerase chain reaction (PCR) with genomic DNA extracted from cultured trophozoites in all four-nuclei, amoeba-positive samples from a population living in Macaparana in northeastern Brazil. Among 1,437 stool samples analyzed, only 59 (4.1%) were positive for four nuclei amoeba. However, all of these samples were negative in an immunoenzymatic assay for the presence of E. histolytica-specific galactose adhesin. Of 59 cultivated samples, only 31 showed trophozoites. Extraction of DNA from these 31 samples, followed by the PCR, showed that 23 samples (74.19%) were positive for E. dispar and no amplification was observed for pathogenic E. histolytica. The remaining eight samples were negative for both species. These findings are consistent with those previously reported.

INTRODUCTION

The protozoa Entamoeba histolytica is an intestinal parasite infecting 500 million people worldwide.1 Up to 100,000 deaths per year have been attributed to complications of amebiasis, notably amoebic liver abscess.2 The prevalence of E. histolytica in developing countries is often assumed to be high, frequently without supporting data.3 Studies on E. histolytica carried out at the Laboratório de Imunopatologia Keizo Asami in Recife between 1988 and 1994 among low-income populations have shown differences in the prevalence of this organism in the northern, northeastern, southeastern regions of Brazil. The methodology used in these studies has ranged from traditional serologic tests, such as gel diffusion precipitin (GDP) and zymodemes, to molecular biology techniques such as restriction endonuclease digestion of amplified genomic DNA.3–7 These investigations showed the presence of both E. histolytica and E. dispar in the Amazon region (northern area) with a higher prevalence of E. histolytica, while E. dispar predominated in the northeastern region.

In contrast to these findings, E. histolytica has been reported in a community in Fortaleza in northeastern Brazil.8 The investigators detected the presence of serum antibodies specific for the Gal/GalNAc lectin of E. histolytica and suggested that this community was highly endemic for E. histolytica with infections rate similar to other developing nations. Despite this result, which differs from those obtained at the Laboratório de Imunopatologia Keizo Asami, the northeastern region seems to have a diverging parasitologic profile with regard to the presence of E. histolytica and E. dispar.

In recent years, a number of methods have been developed for the clear distinction of these two species. Immunoassays have been widely used in routine laboratory analysis. The gel diffusion precipitation test is considered by some researchers to be one of the most reliable serologic tests for the diagnosis of amebiasis.9,10 The enzyme-linked immunosorbent assay (ELISA) is also used in serodiagnosis method. However, this method cannot differentiate between a current and previous parasite infection, and is of limited value when examining individuals from endemic areas with high levels of circulating antibodies.11 Many antigens have been reported as being specific for the diagnosis of amebiasis. These include E. histolytica trophozoite antigens (HM-1 IMSS), pathogen-specific epitopes of the galactose adhesin of E. histolytica, single recombinant E. histolytica antigen (P1-EIA), and the 170-kD antigenic subunit of the amebal Gal/GalNAc-lectin.12–15 Although the use of a stool ELISA has been shown to be useful in routine diagnostic procedures, a comparative study on the use of this ELISA and a polymerase chain reaction (PCR) for the detection of E. histolytica and E. dispar indicated that the PCR was more useful than the ELISA.16

Conversely, a number of DNA sequences have been used as targets for specific detection of E. histolytica using PCR technology. Ribosomal RNA molecules are the most commonly used targets, followed by restriction pattern analysis.16,17–19 In addition, genomic DNA has also been used in a diagnostic PCR.20–23 The primers specific for E. histolytica and E. dispar (P11 plus P12 and P13 plus P14, respectively) were found to show a sensitivity of 100%.24,25 The PCR technique is fast, safe, and constitutes an good approach to obtain information about the occurrence of E. histolytica or E. dispar in the northeastern region of Brazil.

The purpose of this study was to determine the prevalence of E. histolytica and E. dispar by using E. histolytica stool antigen detection kits and a PCR with genomic DNA extracted from cultured trophozoites in a population located in Pernambuco State in northeastern Brazil.

MATERIALS AND METHODS

Samples.

Aliquots of fresh, unpreserved stool obtained from 1,437 randomly selected individuals living in Macaparana, Brazil were stored at 4°C. A one-gram portion was stored at −20°C for subsequent immunoenzymatic analysis. Macaparana is located in Pernambuco State, Brazil, on the limits of a sugarcane plantation area, 118 km from Recife (the capital of Pernambuco). It has a population of 22,494 inhabitants (13,518 and 8,976 in the urban and rural areas, respectively) occupying an area of 103 km2. Illiteracy is very high (65.1%) among the population more than 10 years old. Young people represent most of the population (46.5% of the population is less than or equal to 20 years old and 75% is less than 42 years old). The estimated family income is approximately U.S. $480 per year. The study protocol was reviewed and approved by the Hospital das Clinicas Ethics Committee of the Universidade Federal de Pernambuco, Brazil, and informed consent was obtained from each individual or responsible guardian.

Microscopy analysis.

The presence of parasites in the samples was determined by various concentration methods.26,27

Immunoenzymatic assay.

The presence of E. histolytica-specific galactose adhesin was determined among the samples that were stored at −20°C and positive for the presence of four-nuclei amoeba with a commercially available kit (ELISA kit E. histolytica-II; Techlab, Inc., Blacksburg, VA). This kit is based on the monoclonal antibody-peroxidase conjugate specific for E. histolytica adhesin. According to the manufacturer’s instructions, a positive result was defined as an optical density reading > 0.05 after subtraction of the negative control optical density.

Extraction of genomic DNA.

All four-nuclei, amoeba-positive samples were incubated with the Robinson’s medium at 37°C for 48 hours.28 Cultured trophozoites were centrifuged and resuspended in ethanol. Subsequently, trophozoites were centrifuged and resuspended in 200 μL of the solubilizing agent (10 mM Tris-HCl, pH 7.5, 10 mM EDTA, 0.5% sodium dodecyl sulfate, and 0.5 mg of proteinase K) for two hours at 60°C. Genomic DNA was extracted with phenolchloroform, precipitated with ethanol and 3 M sodium acetate, resuspended in TE buffer (0.01M Tris-HCl, pH 7.4, 2.5 mM EDTA) and stored at −20°C until PCR amplification.

Polymerase chain reaction.

The PCR was conducted in a volume of 25 μL containing (final concentrations) 20 mM Tris-HCl, pH 8.4, 3.0 mM MgCl2, 50 mM KCl, 2.0 mM each of the four dNTPs, 10 pmol of each specific primer (p11 plus p12 and p13 plus p14), 2.0 units of Taq polymerase (Invitrogen, Carlsbad, CA), and approximately 50 ng of genomic DNA. The thermal cycles consisted of an initial denaturation at 94°C for one minute, followed by 30 cycles at 94°C for one minute, 59°C for 90 seconds, 72°C for 90 seconds, and a final extension at 72°C for five minutes. The PCR products were isolated by electrophoresis on 2% agarose gels containing ethidium bromide and the gels were photographed under ultraviolet light. Two DNA samples testing positive for each species were used as positive controls.

RESULTS

Among 1,437 stool samples analyzed by optical microscopy, only 59 (4.1%) were positive for the presence of four-nuclei amoeba, namely, E. histolytica or E. dispar. However, all 59 samples were negative in the immunoenzymatic assay for the presence of E. histolytica-specific galactose adhesin. Microscopic analysis also showed the presence of the following other microorganisms: Entamoeba coli (27), Ascaris lumbricoides and Entamoeba coli (4), Ascaris lumbricoides (3), Entamoeba coli and Endolimax nana (3), Iodameba bütschlii (2), Trichuris trichiura (1), Endolimax nana (1), Ancilostomídeos (1), A. lumbricoides and T. trichiura (1), A. lumbricoides and Enterobius vermicularis (1), A. lumbricoides, I. bütschlii, and Entamoeba coli (1), Entamoeba coli, Ancilostomídeos, and I. bütschlii (1), Entamoeba coli and Schistosoma mansoni (1), I. bütschlii and Giardia lamblia (1), I. bütschlii and Entamoeba coli (1), and no other parasites (10).

Of 59 cultivated samples positive for the presence of four nuclei, only 31 showed trophozoites. This was expected because there are reports describing the impractical and time-consuming nature of obtaining cultures from a large number of microscopy-positive samples.29,30

Extraction of DNA from 31 samples, followed by the PCR, showed that 23 samples (74.19%) were positive for E. dispar as demonstrated by amplification of the species-specific fragment (100 basepairs). Conversely, no amplification was observed for pathogenic E. histolytica (Figure 1). The remaining eight samples were negative for both species. The absence of amplification of these samples indicates either the presence of PCR inhibitors in the stool samples or DNA from trophozoites of Entamoeba species other than E. dispar or E. histolytica.

DISCUSSION

These findings are consistent with those previously reported for the Pernambuco State of Brazil.4,6,7,31,32 They showed a high incidence of four-nuclei Entamoeba and the presence of E. dispar (non-pathogenic amoeba) in this population. Furthermore, the E. histolytica-specific ELISA was shown to be a sensitive and specific method for the rapid differentiation of the two species because its results were comparable to those obtained with the PCR.

These results indicate that in communities in northeastern Brazil, the presence of either four-nuclei amoeba or trophozoites in the stool of a patient with diarrhea is not equal to amebiasis (the presence of the pathogenic E. histolytica). This is important when one considers the currently available treatments, some of which have undesirable side effects. A diagnosis of amebiasis should also be considered when red blood cells are observed within trophozoites in stool specimens.2 We recommend that ELISA procedures based on reliable antigens or antibodies be used in this region. Unfortunately, PCR methods are still too sophisticated and expensive for the public health systems of these communities.

Figure 1.
Figure 1.

Typical polymerase chain reaction amplifications of DNA from trophozoites harvested from 10 stool samples (2–6 and 9–13) and using primers for Entamoeba histolytica (p11/p12) and E. dispar (p13/p14). Lanes a and b = amplifications for the primers p13/p14 (≃ 100 basepairs) and p11/p12, respectively. Lanes 1 and 8 = positive controls for E. dispar; lanes 7 and 14 = positive controls for E. histolytica; lanes M = molecular mass marker.

Citation: The American Journal of Tropical Medicine and Hygiene Am J Trop Med Hyg 70, 2; 10.4269/ajtmh.2004.70.221

Authors’ addresses: Sandra M. B. Pinheiro, Ivanise S. Aca, and João I. Irmão, Departamento de Medicina Tropical, Universidade Federal de Pernambuco, Campus Universitário, 50670-901, Recife, Pernambuco, Brazil. Rosa M. Carneiro, Departamento de Medicina Social, Universidade Federal de Pernambuco, Campus Universitário, 50670-901, Recife, Pernambuco, Brazil. Marcos A. Morais Jr., Departamento de Genética, Universidade Federal de Pernambuco, Campus Universitário, 50670-901, Recife, Pernambuco, Brazil. Maria R. M. Coimbra, Departamento de Pesca, Dom Manoel de Medeiros, Dois Irmãos, 52171-900, Universidade Federal Rural de Pernambuco, Recife, Brazil. Luiz B. Carvalho Jr., Departamento de Bioquímica, Laboratório de Imunopatologia Keizo Asami, Universidade Federal de Pernambuco, Campus Universitário, 50670-901, Recife, Pernambuco, Brazil.

Acknowledgments: We thank Dr. Tsutomo Takeuchi (Department of Tropical Medicine and Parasitology of Keio University, Tokyo, Japan) for providing the primers and positive DNA controls. We also thank Dr. Seiki Kobayashi for helpful discussions during the development of this project.

Financial support: This work was supported by the Conselho Nacional de Desenvolvimento Cientifico e Technológico/Fundo Setorial do Petróleo e Gás Natural (grant number 463655/001), the Fundação de Amparo à Ciéncia e Tecnologia do Estado de Pernambuco (grant number 23-CBIO-08/00-01/01-6), and the Japan International Cooperation Agency.

REFERENCES

  • 1

    Walsh JA, 1986. Problems in recognition and diagnosis of amoebiasis: estimation of the global magnitude of morbidity and mortality. Rev Infect Dis 8 :228–238.

    • Search Google Scholar
    • Export Citation
  • 2

    World Health Organization, 1997. Amoebiasis. Report on the WHO/Pan American Health Organization/ UNESCO Expert Consultation, Mexico City. Wkly Epidemiol Rec 72 :97–100.

    • Search Google Scholar
    • Export Citation
  • 3

    Okazaki M, Miranda P, Neto J, Diegues V, Alves J, Cauas M, Tanabe M, Kobayashi S, Tateno S, Takeuchi T, 1988. Parasitological and serological studies on amoebiasis and other intestinal parasitic infections in Recife and its suburban area, northeast Brazil. Rev Inst Med Trop Sao Paulo 30 :313–321.

    • Search Google Scholar
    • Export Citation
  • 4

    Nozaki T, Aca IS, Okuzawa E, Magalhães M, Tateno S, Takeuchi T, 1990. Zymodemes of Entamoeba histolytica isolated in the Amazon and the northeast of Brazil. Trans R Soc Trop Med Hyg 84 :387–388.

    • Search Google Scholar
    • Export Citation
  • 5

    Aca IS, França JR, Nozaki T, Freitas GB, Tateno S, 1993. Entamoeba histolytica zymodemes in children of Osasco. Rev Inst Med Trop Sao Paulo 35 :581–582.

    • Search Google Scholar
    • Export Citation
  • 6

    Aca IS, Kobayashi S, Carvalho LB Jr, Tateno S, Takeuchi T, 1994. Prevalence and pathogenicity of Entamoeba histolytica in three different regions of Pernambuco. Rev Inst Med Trop Sao Paulo 36 :519–524.

    • Search Google Scholar
    • Export Citation
  • 7

    Tachibana H, Kobayashi S, Paz KC, Aca IS, Tateno S, Ihara S, 1992b. Analysis of pathogenicity by restriction-endonuclease digestion of amplified genomic DNA of Entamoeba histolytica isolated in Pernambuco, Brazil. Parasitol Res 78 :433–436.

    • Search Google Scholar
    • Export Citation
  • 8

    Braga LL, Lima AL, Sears CL, Newman RD, Wuhib T, Paiva CA, Guerrant RL, Mann BJ, 1996. Seroepidemiology of Entamoeba histolytica in a slum in northeast Brazil. Rev Inst Med Trop Sao Paulo 55 :693–697.

    • Search Google Scholar
    • Export Citation
  • 9

    Maddison SE, 1965. Characterization of Entamoeba histolytica antigen-antibody reaction by gel diffusion. Exp Parasitol 16 :224–235.

  • 10

    Patterson M, Healy GR, Shabot JM, 1980. Serologic testing for amoebiasis. Gastroenterology 78 :136–141.

  • 11

    Singh B, 1997. Molecular methods for diagnosis and epidemiological studies of parasitic infections. Int J Parasitol 27 :1135–1145.

  • 12

    Wonsit R, Thammapalerd N, Tharavanij S, Radomyos P, Bunnag D, 1992. Enzyme-linked immunosorbent assay based on monoclonal and polyclonal antibodies for the detection of Entamoeba histolytica antigens in faecal specimens. Trans R Soc Trop Med Hyg 86 :166–169.

    • Search Google Scholar
    • Export Citation
  • 13

    Mackenstedt U, Johnson AM, 1995. Genetic differentiation of pathogenic and non-pathogenic strains of Entamoeba histolytica by random amplified polymorphic DNA-polymerase chain reaction. Parasitol Res 81 :217–221.

    • Search Google Scholar
    • Export Citation
  • 14

    Haque R, Kress K, Wood S, Jackson TFHG, Lyerly D, Petri WA Jr, 1993. 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.

    • Search Google Scholar
    • Export Citation
  • 15

    Shenai BR, Komalam BL, Arvind AS, 1996. Recombinant antigen-based avidin-biotin microtiter enzyme-linked immunosorbent assay for serodiagnosis of invasive amebiasis. J Clin Microbiol 34 :828–833.

    • Search Google Scholar
    • Export Citation
  • 16

    Mirelman D, Nuchamowitz Y, Stolarsky T, 1997. Comparison of enzyme-linked immunosorbent assay-based kits and PCR amplification of rRNA genes for simultaneous detection of Entamoeba histolytica and E. dispar. J Clin Microbiol 5 :2405–2407.

    • Search Google Scholar
    • Export Citation
  • 17

    Clark CG, Diamond LS, 1991. Ribosomal RNA genes of ‘pathogenic’ and ‘nonpathogenic’ Entamoeba histolytica are distinct. Mol Biochem Parasitol 47 :297–302.

    • Search Google Scholar
    • Export Citation
  • 18

    Novati S, Sironi M, Granata S, Bruno A, Gatti S, Scaglia M, Bandi C, 1996. 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.

    • Search Google Scholar
    • Export Citation
  • 19

    Myjak P, Kur J, Pietkiewicz H, 1997. Usefulness of new DNA extraction procedure for PCR technique in species identification of Entamoeba isolates. Wiad Parazytol 43 :163–170.

    • Search Google Scholar
    • Export Citation
  • 20

    Tachibana H, Kobayashi S, Nagakura K, Kaneda Y, Takeuchi T, 1991. Reactivity of monoclonal antibodies to species-specific antigens of Entamoeba histolytica. J Protozool 38 :329–334.

    • Search Google Scholar
    • Export Citation
  • 21

    Cheng XJ, Tachibana H, Kobayashi S, Kaneda Y, Huang MY, 1993. Pathogenicity of Entamoeba histolytica isolates from Shangai, China. Parasitol Res 79 :608–610.

    • Search Google Scholar
    • Export Citation
  • 22

    Rivera WL, Tachibana H, Silva-Tahat MRA, Haruki U, Kanbara H, 1996. Differentiation of Entamoeba histolytica and E. dispar DNA from cysts present in stool specimens by polymerase chain reaction: its field application in the Philippines. Parasitol Res 82 :585–589.

    • Search Google Scholar
    • Export Citation
  • 23

    Tachibana H, Cheng XJ, Kobayashi S, Matsubayashi N, Goth S, Matsubayashi K, 2001. High prevalence of infection with Entamoeba dispar but not E. histolytica, in captive macaques. Parasitol Res 87 :14–17.

    • Search Google Scholar
    • Export Citation
  • 24

    Tachibana H, Kobayashi S, Takekoshi M, Ihara S, 1991. Distinguishing pathogenic and nonpathogenic isolates of Entamoeba histolytica by polymerase chain reaction. J Infect Dis 164 :825–826.

    • Search Google Scholar
    • Export Citation
  • 25

    Zaman S, Khoo J, Ng SW, Ahmed R, Khan MA, Hussain R, Zaman V, 2000. Direct amplification of Entamoeba histolytica DNA from amoebic liver abscess pus using polymerase chain reaction. Parasitol Res 86 :101–109.

    • Search Google Scholar
    • Export Citation
  • 26

    Hoffman WA, Pons JA, Janer JL, 1934. The sedimentation concentration method in schistosomiasis mansoni. J Public Health Trop Med 9 :283–298.

    • Search Google Scholar
    • Export Citation
  • 27

    Ritchie LS, 1948. An ether sedimentation technique for routine stool examination. Bull USA Med Dept 8 :326.

  • 28

    Robinson GI, 1968. The laboratory diagnosis of human parasitic amoeba. Trans R Soc Trop Med Hyg 62 :285–293.

  • 29

    Haque R, Neville LM, Hahn P, Petri WA Jr, 1995. Rapid diagnosis of Entamoeba infection by using Entamoeba and Entamoeba histolytica stool antigen detection kits. J Clin Microbiol 33 :2558–2561.

    • Search Google Scholar
    • Export Citation
  • 30

    Sehgal R, Abd-Alla M, Moody AH, Chiodini PL, Ackers JP, 1995. Comparison of two media for the isolation and short-term culture of Entamoeba histolytica and E. dispar. Trans R Soc Trop Med Hyg 89 :394.

    • Search Google Scholar
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Author Notes

Reprint requests: Luiz B. Carvalho Jr., Laboratório de Imunopatologia Keizo Asami, Universidade Federal de Pernambuco, Campus Universitário, 50670-901, Recife, Pernambuco, Brazil, Telephone: 55-81-3271-8484, Fax: 55-81-3271-8485, E-mail: lbcj@lika.ufpe.br.
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