Parasitol Res (1992) 78:433~436
Parasitnlngy Research 9 Springer-Verlag1992
Analysis of pathogenicity by restriction-endonuclease digestion of amplified genomic DNA of Entamoeba histolytica isolated in Pernambuco, Brazil* Hiroshi Tachibana 1' 2, Seiki Kobayashi 1' 3, Kilma C. Paz 1, Ivanize S. Aca 1, Seiki Tateno 1, and Seiji Ihara 4 1 Laborat6rio de Imunopatologia Prof. Keizo Asami, Universidade Federal de Pernambuco, Recife, Pernambuco, Brazil 2 Department of Parasitology and 4 Department of Molecular Biology, Tokai University School of Medicine, Bohseidai, Isehara, Kanagawa 259-11, Japan 3 Department of Parasitology, School of Medicine, Keio University, 35 Shinanomachi, Shinjuku-ku, Tokyo 160, Japan Accepted March 1, 1992
Abstract. The pathogenicity of 47 strains of Entamoeba histolytica isolated in Pernambuco, Brazil, was examined using the polymerase chain reaction (PCR) followed by restriction-endonuclease digestion. Electrophoretic patterns of PCR products digested with HinfI revealed that all strains were nonpathogenic. The results were entirely in accord with phenotypic properties such as isoenzyme patterns and the failure to bind a pathogenic-isolatespecific monoclonal antibody. When the sensitivity of PCR was examined, amplified products could be detected from template D N A equivalent to five trophozoites. These observations indicate that P C R amplification of genomic D N A and subsequent restriction-enzyme digestion is a useful strategy for obtaining a sensitive and accurate diagnosis. The present study also demonstrates that nonpathogenic strains of E. histolytica predominate in northeastern Brazil.
The protozoan parasite Entamoeba histolytica infects approximately 500 million people, or one-tenth of the world's population. O f these, 40 million develop hemorrhagic colitis and extraintestinal abscesses, whereas the rest generally remain symptom-free (Walsh 1988), primarily due to the lack of pathogenicity of many amoebic isolates. Pathogenic isolates can be distinguished by their isoenzyme electrophoretic patterns (zymodemes; Sargeaunt 1988). In addition, recent advances in molecular biology have revealed genomic D N A differences between pathogenic and nonpathogenic isolates of E. histolytica (Garfinkel et al. 1989; Tannich et al. 1989, 1991; Bracha et al. 1990; Edman et al. 1990). * This study was supported by the Japan International Cooperation Agency (JICA), by a Tokai University School of Medicine Research Aid award, and by a grant from the Ohyama Health Foundation Correspondence to: H. Tachibana, Department of Parasitology, Tokai University School of Medicine, Bohseidai, Isehara, Kanagawa 259-11, Japan
The cloning and sequencing of the c D N A coding the 30-kDa antigen of E. histolytica, which is identified by the pathogenic-isolate-specifc monoclonal antibody (mAb 4G6), has also been reported (Tachibana et al. 1990, 1991a, b). Direct sequencing of genomic D N A amplified by a polymerase chain reaction (PCR; Saiki et al. 1988) has revealed a 5% difference in the nucleotide sequences of pathogenic and nonpathogenic isolates. Moreover, it has been shown that the combined use of P C R and restriction-endonuclease digestion is useful for the identification of species and the determination of their pathogenicity, although the number of strains examined were limited (Tachibana et al. 1991 a). In northeastern Brazil, where diarrhoeal diseases are common, a high prevalence of E. histolytica has been reported; however, serologic studies and zymodeme analysis of isolates demonstrated the presence of many nonpathogenic strains (Okazaki et al. 1988; Gongalves et al. 1990; Nozaki et al. 1990). The present study was therefore undertaken to determine whether the E. histoIytica strains isolated in Pernambuco are genotypically nonpathogenic. The value of restriction-enzyme digestion of PCR-amplified products as a diagnostic tool was also evaluated.
Materials and methods
Entamoeba histolytica isolates Stool samples containing E. histolytieawere collected from inhabitants in five locations in Pernambuco State: Recife (Capital of Pernambuco), JaboatS~o (15 km west of Recife), G16ria do Goitfi (50 km west of Recife), Palmares (100 km southwest of Recife), and Bodoc6 (560 km west of Recife). The samples were inoculated into Robinson's medium (Robinson 1968) and cultured at 37~ C. Zymodemes of the isolates were determined according to the procedure of Sargeaunt et al. (1984). The reactivity of mAb 4G6 with trophozoites of E. histolytica isolates was examined by an indirect fluorescence antibody (IFA) test as previously described by Tachibana et al. (1990). The SAW408 and SAW142 strains of E. histolytica, kindly provided by Dr. P.G. Sargeaunt, were used as pathogenic and nonpathogenic controls, respectively. Serologic examina-
434 tion of the donors was carried out by the gel-diffusion precipitin test as previously described by Takeuchi and Kobayashi (1983).
M
1
2
3
4
5
6
7
8
910
A
Preparation of DNA samples E. histolytica trophozoites grown in Robinson's medium were isolated with Percoll and then lysed by shaking for 2 h at 60~ in lysis buffer [100 mM NaC1, 10 mM TRIS, 10 mM ethylenediaminetetraacetic acid (EDTA), 0.5% sodium N-lauroyl sarcosinate, and 0.5 mg proteinase K/ml]. The DNA was extracted with phenol/ chloroform/isoamyl alcohol and ethanol-precipitated with sodium acetate (Tachibana et al. 1991a).
100 bp-
E
Polymerase chain reaction 100
DNA amplification was carried out using primers pl (5'TAAAGCACCAGCATATTGTC3') plus p4 (5'TTAATTCCATCTGGT GTTGG3') as described elsewhere (Tachibana et al. 1991a). The 30-cycle PCR consisted of denaturation (94~ C) for 1 rain (3 rain in cycle 1), annealing (55~ C) for 2 rain, and polymerization (72~ C) for 2 rain (9 rain in cycle 30).
Restriction-endonuclease digestion A 10-gl portion of the amplified products was subjected to HinfI and EcoT22I digestion for 1 h at 37~ C and then electrophoresed in 3% Nusieve-1% agarose gels containing ethidium bromide. The presence of specific bands was visualized under ultraviolet light.
Fig. 1A, B. Electrophoretic separation of PCR products digested with A Hint'[ and B EcoT22I. Template DNA from various isolates of Entamoeba histolytica was amplified for 30 cycles using pl plus p4 primers. Amplified products (10 gl) were digested and then subjected to electrophoresis in 3% NuSieve-1% agarose gel containing ethidium bromide. Lane 1, SAW408 isolate (pathogenic control); lane 2, SAWI42 isolate (nonpathogenic control); lane 3, R-6003 isolate [zymodeme (Z), Z-I]; lane 4, R-5008 isolate (Z-III); lane 5, B-106 isolate (Z-IV); lane 6, R-4006 isolate (Z-VIII); lane 7, P-84 isolate (Z-IX); lane 8, JB-13 isolate (Z-X); lane 9, R-5029 isolate (Z-XVII); lane iO, JB-26 isolate (Z-XVIII); M, DNA size marker (100-bp ladder)
Results and discussion
When D N A extracted from Entamoeba histolytica isolates was amplified by PCR for 30 cycles, 531-bp products were obtained from all 47 strains. HinfI digestion of these PCR products consistently yielded the same three fragments (Fig. 1 A, Table 1). The digestion patterns obtained were identical to that of the nonpathogenic SAW142 control strain and differed from that of the pathogenic SAW408 strain. On the other hand, treatment with EcoT22I did not affect the PCR products of the 47 E. histolytica isolates, whereas the PCR product from the SAW408 strain was split into 2 fragments by EcoT22I, as previously reported (Fig. 1 B). These observations indicate that all 47 strains of E. histolytica isolated in Brazil are genotypically nonpathogenic. These results correlate completely with those obtained using zymodeme analysis (Table 1). In the 125-kDa antigen gene of E. histolytiea, a 10% difference in the nucleotide sequences of the pathogenic and nonpathogenic isolates has recently been demonstrated (Tannich etal. 1989; Edman etal. 1990). Although endonuclease digestion of the PCR-amplified products of the genomic D N A yielded restriction fragments characteristic of pathogenic and nonpathogenic isolates, rare exceptions have been observed (Edman et al. 1990; Tannich and Burchard 1991). In a previous study, three nonpathogenic isolates possessing two types of zymodemes were examined by P C R and restrictionenzyme digestion (Tachibana et al. 1991a). As shown in Table 1, the 47 Brazilian E. histolytica isolates used
in the present study demonstrated 8 types of nonpathogenic zymodeme patterns, amounting to two-thirds of the known nonpathogenic zymodemes. Therefore, the present observations strongly support the concept that pathogenic and nonpathogenic isolates are genomically different with respect to the gene encoding the 30-kDa antigen. Furthermore, the nucleotide sequences of the amplified parts of genes seem to be well conserved among the pathogenic and nonpathogenic isolates, respectively, although a strain-specific D N A has also been identified (Burch et al. 1991). In the present study, ten strains were isolated from individuals presenting wih diarrhea and/or bloody stools (Table 1). Nevertheless, the phenotypic and genotypic properties of the isolates indicated that they were nonpathogenic. The observation that antibodies against E. histolytica were not detected in any of the serum samples also indicated that the isolates were noninvasive (Table 1). Therefore, the variety of symptoms observed in the infected individuals seems to have been caused by other factors. Indeed, a high prevalence of other parasites such as Giardia lamblia, Strongyloides stercoralis, and Schistosoma mansoni, which also cause bowel disease, diarrhea, and dysentery, has been reported in northeastern Brazil (Okazaki etal. 1988; Gongalves et al. 1990; Tanabe et al. 1990). Although it cannot be concluded with certainty that pathogenic isolates do not exist in the survey area, the present study demonstrates that nonpathogenic E. histolytica strains predominate in northeastern Brazil.
435 Table 1. Characteristics of individuals infected with
Entamoeba histolytica and the pathogenicity of the isolates studied
Isolates
Geographic origin
Symptoms
Serology
Zymodeme
Reactivity to mAb 4G6
Hinfi digests of PCR products
B-5 B-40 B- 106 B- 125 B-I 31 B-136 B-147 B-196 G-3 G-62 G-68 G-77 G-93 JB-3 JB-5 JB-8 JB- 13 JB-26 JB-29 L-22 P-2 P-6 P-8 P-10 P-I 3 P-14 P-17 P-20 P-23 P-36 P-84 R-2003 R-3002 R-3080 R-4006 R-4007 R-4009 R-4019 R-4025 R-4044 R-5008 R-5013 R-5029 R-6003 R-6004 R-6008 T-11
Bodoc6 Bodoc6 Bodoc6 Bodoc6 Bodoc6 Bodoc6 Bodoc6 Bodoc6 G16ria do Gldria do Gldria do Gldria do Gl6ria do Jaboat~.o Jaboatfio Jaboatfio Jaboatfio Jaboatg.o Jaboatfio Recife Palmares Palmares Palmares Palmares Palmares Palmares Palmares Palmares Palmares Palmares Pahnares. Recife Recife Recife Recife Recife Recife Recife Recife Recife Recife Recife Recife Recife Recife Recife Recife
DI DI AP AS AP AS AS AS AS DI AS DI DI AS AS AS AS AS AS AIDS, HS AS AP AP AS AP AN BS, DI AS AP, DI AS AS AS DI AS AS AS AS AS AS AS AS AS AS BS, DI AS AS BS, DI
ND ND ND -
I XVII IV XVII XVIII VIII VIII X XVII I I I I XVII XVII VIII X XVIII XVII XVIII I I I XVII I I XVII XVII III III IX III I XVIII VIII VIII XVIII I I XVII III VIII XVII I XVII XVIII XVIII
-
NP NP NP NP NP NP NP NP NP NP NP NP NP NP NP NP NP NP NP NP NP NP NP NP NP NP NP NP NP NP NP NP NP NP NP NP NP NP NP NP NP NP NP NP NP NP NP
Goitfi Goitfi Goitfi Goitfi Goit/~
ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND -
-
-
-
AIDS, Acquired immunodeficiency syndrome; AN, anemia; AP, abdominal pain; AS, asymptomatic; BS, bloody stool; DI, diarrhea; HS, homosexual; - , negative; NP, nonpathogenic; ND, not done
To e v a l u a t e t h e s e n s i t i v i t y o f t h e P C R a n a l y s i s , D N A from the SAW408 strain was diluted and then amplified f o r 30 cycles. T h e r e s u l t s o f e l e c t r o p h o r e s i s o f o n e - t e n t h o f t h e a m p l i f i e d p r o d u c t s a r e i l l u s t r a t e d i n Fig. 2. W h e n t e m p l a t e D N A e q u i v a l e n t t o > 100 t r o p h o z o i t e s w a s amplified, a distinct band was observed by ethidium bromide staining. A faint band was visible even when genom i c D N A e q u i v a l e n t t o five cells w a s u s e d a s a t e m p l a t e . Such high sensitivity was on a par with that previously r e p o r t e d b y T a n n i c h a n d B u r c h a r d (1991). A l t h o u g h rela t i v e l y l a r g e n u m b e r s o f o r g a n i s m s a r e r e q u i r e d f o r zy-
modeme analysis, smaller numbers are sufficient for PCR analysis and IFA testing with mAbs. S a r g e a u n t (1988) c l a s s i f i e d 22 d i f f e r e n t t y p e s o f z y m o demes on the basis of the electrophoretic patterns of 4 isoenzymes. A more recent study suggests that the a p p e a r a n c e o f so m a n y t y p e s o f z y m o d e m e s d e p e n d e d on the culture conditions used (Blanc and Sargeaunt 1991). H o w e v e r , P C R a n a l y s i s u s i n g E. histolytica-specific p r i m e r s c o u l d m i n i m i z e t h e i n f l u e n c e o f c u l t u r e c o n d i t i o n s a n d / o r o t h e r p a r a s i t i c a m e b a e ( T a c h i b a n a e t al. 1991a). Since the possibility o f c o n v e r s i o n f r o m n o n -
436
M
1
2
3
4
5
6
7
531 bp
Fig. 2. Sensitivity of PCR amplification of genomic DNA from Entamoeba histolytica trophozoites. The DNA extracted from the SAW408 strain was serially diluted and amplified for 30 cycles using pl plus p4 primers. One-tenth of the amplified products was subjected to electrophoresis in 3% NuSieve-1% agarose gel containing ethidium bromide. The number of trophozoites equivalent to the template DNA used as shown in lanes 1-7; 0, 1, 5, 10, 10 2, 10 3, and 104 organisms, respectively; M, HincII-cleaved 9X174. The arrow indicates the position and size of PCR products p a t h o g e n i c i t y to p a t h o g e n i c i t y (or the reverse) c a n n o t be c o m p l e t e l y r u l e d o u t ( M i r e l m a n 1987; A n d r e w s et al. 1990), the g e n o t y p i c i d e n t i f i c a t i o n o f E. histolytica isolates using P C R is r e c o m m e n d e d for a sensitive a n d a c c u r a t e diagnosis.
References Andrews BJ, Mentzoni L, Bjorvatn B (1990) Zymodeme conversion of isolates of Entamoeba histolytica. Trans R Soc Trop Med Hyg 84:63-65 Blanc D, Sargeaunt PG (1991) Entamoeba histolytica zymodemes: exhibition of 7 and 6 bands only of glucose phosphate isomerase and phosphoglucomutase may be influenced by starch, content in the medium. Exp Parasitol 72:87-90 Bracha R, Diamond LS, Ackers JP, Burchard GD, Mirelman D (1990) Differentiation of clinical isolates of Entamoeba histolytica by using specific DNA probes. J Clin Microbiol 28 : 680-684 Burch DJ, Li E, Reed S, Jackson TFHG, Stanley SL Jr (1991) Isolation of a strain-specific Entamoeba histolytica cDNA clone. J Clin Microbiol 29:696-701 Edman U, Meraz MA, Rausser S, Agabian N, Meza I (1990) Char= acterization of an immunodominant variable surface antigen from pathogenic and nonpathogenic Entamoeba histolytiea. J Exp Med 172: 879-888 Garfinkel LI, Giladi M, Huber M, Gitler C, Mirelman D, Revel M, Rozenblatt S (1989) DNA probes specific for Entamoeba histolytica possessing pathogenic and nonpathogenic zymodemes. Infect Immun 57:926-931 Gongalves JF, Tanabe M, Medeiros FPM, Gongalves F J, Aca IS, Motta SRN, Tateno S, Takeuchi T (1990) Parasitological and serological studies on amoebiasis and other intestinal parasitic infections in the rural sector around Recife, northeast Brazil. Rev Inst Med Trop Sao Paulo 32:428-435
Mirelman D (1987) Effect of culture conditions and bacterial associates on the zymodemes of Entamoeba histolytiea. Parasitol Today 3 : 37-40 Nozaki T, Aca IS, Okuzawa E, Magalhges M, Tateno S, Takeuchi T (1990) Zymodemes of Entamoeba histolytica isolated in the Amazon and the north-east of Brazil. Trans R Soc Trop Med Hyg 84:387 388 Okazaki M, Miranda P, Neto J, Diegues V, Alves J, Cauas M, Tanabe M, Kobayashi S, Kaneko N, Nagakura K, Kobayashi M, Motta S, Tateno S, Takeuchi T (1988) Parasitological and serological studies on amoebiasis and other intestinal parasitic infection in Recife and its suburban area, northeast Brazil. Rev Inst Med Trop Sao Paulo 30:313 321 Robinson GL (1968) The laboratory diagnosis of human parasitic amoebae. Trans R Soc Trop Med Hyg 62:285-294 Saiki RK, Gelfand DH, Stoffel S, Scharf SJ, Higuchi R, Horn GT, Mullis KB, Erlich HA (1988) Primer-directed enzyme amplification of DNA with a thermostable DNA polymerase. Science 239:487-491 Sargeaunt PG (1988) Zymodemes of Entamoeba histolytiea. In: Ravdin JI (ed) Amebiasis: human infection by Entamoeba histolytiea. John Wiley & Sons, New York, pp 370-387 Sargeaunt PG, Baveja UK, Nanda R, Anand BS (1984) Influence of geographical factors in the distribution of pathogenic zymodemes of Entamoeba histolytica: identification of zymodeme XIV in India. Trans R Soc Trop Med Hyg 78:96-101 Tachibana H, Kobayashi S, Kato Y, Nagakura K, Kaneda Y, Takeuchi T (1990) Identification of a pathogenic isolate-specific 30,000-M, antigen of Entamoeba histolytica by using a monoclonal antibody. Infect Immun 58:955-960 Tachibana H, Ihara S, Kobayashi S, Kaneda Y, Takeuchi T, Watanabe Y (1991a) Differences in genomic DNA sequences between pathogenic and nonpathogenic isolates of Entamoeba histolytiea identified by polymerase chain reaction. J Clin Microbiol 29 : 2234-2239 Tachibana H, Kobayashi S, Nagakura K, Kaneda Y, Takeuchi T (1991 b) Reactivity of monoclonal antibodies to species-specific antigens of Entamoeba histolytiea. J Protozool 38:329-334 Takeuchi T, Kobayashi S (1983) Serological diagnosis of amoebiasis. Immunoadvance 12:27-30 Tanabe M, Okazaki M, Okazaki M, Kobayashi S, Kaneko N, Sekiguchi T, Tateno S, Motta SRN, Takeuchi T (1990) Serological studies on schistosomiasis mansoni in the northeast of Brazil (I). Rev Inst Med Trop Sao Paulo 32:121-131 Tannich E, Burchard GD (1991) Differentiation of pathogenic from nonpathogenic Entamoeba histolytica by restriction fragment analysis of a single gene amplified in vitro. J Clin Microbiol 29:250-255 Tannich E, Horstmann RD, Knobloch J, Arnold HH (1989) Genomic DNA differences between pathogenic and nonpathogenic Entamoeba histolytica. Proc Natl Acad Sci USA 86:5118-5122 Tannich E, Scholze H, Nickel R, Horstmann RD (1991) Homologous cysteine proteinases of pathogenic and nonpathogenic Entamoeba histolytiea. J Biol Chem 266 : 47984803 Walsh JA (1988) Prevalence of Entamoeba histolytica infection. In: Ravdin JI (ed) Amebiasis: human infection by Entamoeba histolytica. John Wiley & Sons, New York, pp 93-105
Parasitnlngy Research 9 Springer-Verlag1992
Analysis of pathogenicity by restriction-endonuclease digestion of amplified genomic DNA of Entamoeba histolytica isolated in Pernambuco, Brazil* Hiroshi Tachibana 1' 2, Seiki Kobayashi 1' 3, Kilma C. Paz 1, Ivanize S. Aca 1, Seiki Tateno 1, and Seiji Ihara 4 1 Laborat6rio de Imunopatologia Prof. Keizo Asami, Universidade Federal de Pernambuco, Recife, Pernambuco, Brazil 2 Department of Parasitology and 4 Department of Molecular Biology, Tokai University School of Medicine, Bohseidai, Isehara, Kanagawa 259-11, Japan 3 Department of Parasitology, School of Medicine, Keio University, 35 Shinanomachi, Shinjuku-ku, Tokyo 160, Japan Accepted March 1, 1992
Abstract. The pathogenicity of 47 strains of Entamoeba histolytica isolated in Pernambuco, Brazil, was examined using the polymerase chain reaction (PCR) followed by restriction-endonuclease digestion. Electrophoretic patterns of PCR products digested with HinfI revealed that all strains were nonpathogenic. The results were entirely in accord with phenotypic properties such as isoenzyme patterns and the failure to bind a pathogenic-isolatespecific monoclonal antibody. When the sensitivity of PCR was examined, amplified products could be detected from template D N A equivalent to five trophozoites. These observations indicate that P C R amplification of genomic D N A and subsequent restriction-enzyme digestion is a useful strategy for obtaining a sensitive and accurate diagnosis. The present study also demonstrates that nonpathogenic strains of E. histolytica predominate in northeastern Brazil.
The protozoan parasite Entamoeba histolytica infects approximately 500 million people, or one-tenth of the world's population. O f these, 40 million develop hemorrhagic colitis and extraintestinal abscesses, whereas the rest generally remain symptom-free (Walsh 1988), primarily due to the lack of pathogenicity of many amoebic isolates. Pathogenic isolates can be distinguished by their isoenzyme electrophoretic patterns (zymodemes; Sargeaunt 1988). In addition, recent advances in molecular biology have revealed genomic D N A differences between pathogenic and nonpathogenic isolates of E. histolytica (Garfinkel et al. 1989; Tannich et al. 1989, 1991; Bracha et al. 1990; Edman et al. 1990). * This study was supported by the Japan International Cooperation Agency (JICA), by a Tokai University School of Medicine Research Aid award, and by a grant from the Ohyama Health Foundation Correspondence to: H. Tachibana, Department of Parasitology, Tokai University School of Medicine, Bohseidai, Isehara, Kanagawa 259-11, Japan
The cloning and sequencing of the c D N A coding the 30-kDa antigen of E. histolytica, which is identified by the pathogenic-isolate-specifc monoclonal antibody (mAb 4G6), has also been reported (Tachibana et al. 1990, 1991a, b). Direct sequencing of genomic D N A amplified by a polymerase chain reaction (PCR; Saiki et al. 1988) has revealed a 5% difference in the nucleotide sequences of pathogenic and nonpathogenic isolates. Moreover, it has been shown that the combined use of P C R and restriction-endonuclease digestion is useful for the identification of species and the determination of their pathogenicity, although the number of strains examined were limited (Tachibana et al. 1991 a). In northeastern Brazil, where diarrhoeal diseases are common, a high prevalence of E. histolytica has been reported; however, serologic studies and zymodeme analysis of isolates demonstrated the presence of many nonpathogenic strains (Okazaki et al. 1988; Gongalves et al. 1990; Nozaki et al. 1990). The present study was therefore undertaken to determine whether the E. histoIytica strains isolated in Pernambuco are genotypically nonpathogenic. The value of restriction-enzyme digestion of PCR-amplified products as a diagnostic tool was also evaluated.
Materials and methods
Entamoeba histolytica isolates Stool samples containing E. histolytieawere collected from inhabitants in five locations in Pernambuco State: Recife (Capital of Pernambuco), JaboatS~o (15 km west of Recife), G16ria do Goitfi (50 km west of Recife), Palmares (100 km southwest of Recife), and Bodoc6 (560 km west of Recife). The samples were inoculated into Robinson's medium (Robinson 1968) and cultured at 37~ C. Zymodemes of the isolates were determined according to the procedure of Sargeaunt et al. (1984). The reactivity of mAb 4G6 with trophozoites of E. histolytica isolates was examined by an indirect fluorescence antibody (IFA) test as previously described by Tachibana et al. (1990). The SAW408 and SAW142 strains of E. histolytica, kindly provided by Dr. P.G. Sargeaunt, were used as pathogenic and nonpathogenic controls, respectively. Serologic examina-
434 tion of the donors was carried out by the gel-diffusion precipitin test as previously described by Takeuchi and Kobayashi (1983).
M
1
2
3
4
5
6
7
8
910
A
Preparation of DNA samples E. histolytica trophozoites grown in Robinson's medium were isolated with Percoll and then lysed by shaking for 2 h at 60~ in lysis buffer [100 mM NaC1, 10 mM TRIS, 10 mM ethylenediaminetetraacetic acid (EDTA), 0.5% sodium N-lauroyl sarcosinate, and 0.5 mg proteinase K/ml]. The DNA was extracted with phenol/ chloroform/isoamyl alcohol and ethanol-precipitated with sodium acetate (Tachibana et al. 1991a).
100 bp-
E
Polymerase chain reaction 100
DNA amplification was carried out using primers pl (5'TAAAGCACCAGCATATTGTC3') plus p4 (5'TTAATTCCATCTGGT GTTGG3') as described elsewhere (Tachibana et al. 1991a). The 30-cycle PCR consisted of denaturation (94~ C) for 1 rain (3 rain in cycle 1), annealing (55~ C) for 2 rain, and polymerization (72~ C) for 2 rain (9 rain in cycle 30).
Restriction-endonuclease digestion A 10-gl portion of the amplified products was subjected to HinfI and EcoT22I digestion for 1 h at 37~ C and then electrophoresed in 3% Nusieve-1% agarose gels containing ethidium bromide. The presence of specific bands was visualized under ultraviolet light.
Fig. 1A, B. Electrophoretic separation of PCR products digested with A Hint'[ and B EcoT22I. Template DNA from various isolates of Entamoeba histolytica was amplified for 30 cycles using pl plus p4 primers. Amplified products (10 gl) were digested and then subjected to electrophoresis in 3% NuSieve-1% agarose gel containing ethidium bromide. Lane 1, SAW408 isolate (pathogenic control); lane 2, SAWI42 isolate (nonpathogenic control); lane 3, R-6003 isolate [zymodeme (Z), Z-I]; lane 4, R-5008 isolate (Z-III); lane 5, B-106 isolate (Z-IV); lane 6, R-4006 isolate (Z-VIII); lane 7, P-84 isolate (Z-IX); lane 8, JB-13 isolate (Z-X); lane 9, R-5029 isolate (Z-XVII); lane iO, JB-26 isolate (Z-XVIII); M, DNA size marker (100-bp ladder)
Results and discussion
When D N A extracted from Entamoeba histolytica isolates was amplified by PCR for 30 cycles, 531-bp products were obtained from all 47 strains. HinfI digestion of these PCR products consistently yielded the same three fragments (Fig. 1 A, Table 1). The digestion patterns obtained were identical to that of the nonpathogenic SAW142 control strain and differed from that of the pathogenic SAW408 strain. On the other hand, treatment with EcoT22I did not affect the PCR products of the 47 E. histolytica isolates, whereas the PCR product from the SAW408 strain was split into 2 fragments by EcoT22I, as previously reported (Fig. 1 B). These observations indicate that all 47 strains of E. histolytica isolated in Brazil are genotypically nonpathogenic. These results correlate completely with those obtained using zymodeme analysis (Table 1). In the 125-kDa antigen gene of E. histolytiea, a 10% difference in the nucleotide sequences of the pathogenic and nonpathogenic isolates has recently been demonstrated (Tannich etal. 1989; Edman etal. 1990). Although endonuclease digestion of the PCR-amplified products of the genomic D N A yielded restriction fragments characteristic of pathogenic and nonpathogenic isolates, rare exceptions have been observed (Edman et al. 1990; Tannich and Burchard 1991). In a previous study, three nonpathogenic isolates possessing two types of zymodemes were examined by P C R and restrictionenzyme digestion (Tachibana et al. 1991a). As shown in Table 1, the 47 Brazilian E. histolytica isolates used
in the present study demonstrated 8 types of nonpathogenic zymodeme patterns, amounting to two-thirds of the known nonpathogenic zymodemes. Therefore, the present observations strongly support the concept that pathogenic and nonpathogenic isolates are genomically different with respect to the gene encoding the 30-kDa antigen. Furthermore, the nucleotide sequences of the amplified parts of genes seem to be well conserved among the pathogenic and nonpathogenic isolates, respectively, although a strain-specific D N A has also been identified (Burch et al. 1991). In the present study, ten strains were isolated from individuals presenting wih diarrhea and/or bloody stools (Table 1). Nevertheless, the phenotypic and genotypic properties of the isolates indicated that they were nonpathogenic. The observation that antibodies against E. histolytica were not detected in any of the serum samples also indicated that the isolates were noninvasive (Table 1). Therefore, the variety of symptoms observed in the infected individuals seems to have been caused by other factors. Indeed, a high prevalence of other parasites such as Giardia lamblia, Strongyloides stercoralis, and Schistosoma mansoni, which also cause bowel disease, diarrhea, and dysentery, has been reported in northeastern Brazil (Okazaki etal. 1988; Gongalves et al. 1990; Tanabe et al. 1990). Although it cannot be concluded with certainty that pathogenic isolates do not exist in the survey area, the present study demonstrates that nonpathogenic E. histolytica strains predominate in northeastern Brazil.
435 Table 1. Characteristics of individuals infected with
Entamoeba histolytica and the pathogenicity of the isolates studied
Isolates
Geographic origin
Symptoms
Serology
Zymodeme
Reactivity to mAb 4G6
Hinfi digests of PCR products
B-5 B-40 B- 106 B- 125 B-I 31 B-136 B-147 B-196 G-3 G-62 G-68 G-77 G-93 JB-3 JB-5 JB-8 JB- 13 JB-26 JB-29 L-22 P-2 P-6 P-8 P-10 P-I 3 P-14 P-17 P-20 P-23 P-36 P-84 R-2003 R-3002 R-3080 R-4006 R-4007 R-4009 R-4019 R-4025 R-4044 R-5008 R-5013 R-5029 R-6003 R-6004 R-6008 T-11
Bodoc6 Bodoc6 Bodoc6 Bodoc6 Bodoc6 Bodoc6 Bodoc6 Bodoc6 G16ria do Gldria do Gldria do Gldria do Gl6ria do Jaboat~.o Jaboatfio Jaboatfio Jaboatfio Jaboatg.o Jaboatfio Recife Palmares Palmares Palmares Palmares Palmares Palmares Palmares Palmares Palmares Palmares Pahnares. Recife Recife Recife Recife Recife Recife Recife Recife Recife Recife Recife Recife Recife Recife Recife Recife
DI DI AP AS AP AS AS AS AS DI AS DI DI AS AS AS AS AS AS AIDS, HS AS AP AP AS AP AN BS, DI AS AP, DI AS AS AS DI AS AS AS AS AS AS AS AS AS AS BS, DI AS AS BS, DI
ND ND ND -
I XVII IV XVII XVIII VIII VIII X XVII I I I I XVII XVII VIII X XVIII XVII XVIII I I I XVII I I XVII XVII III III IX III I XVIII VIII VIII XVIII I I XVII III VIII XVII I XVII XVIII XVIII
-
NP NP NP NP NP NP NP NP NP NP NP NP NP NP NP NP NP NP NP NP NP NP NP NP NP NP NP NP NP NP NP NP NP NP NP NP NP NP NP NP NP NP NP NP NP NP NP
Goitfi Goitfi Goitfi Goitfi Goit/~
ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND -
-
-
-
AIDS, Acquired immunodeficiency syndrome; AN, anemia; AP, abdominal pain; AS, asymptomatic; BS, bloody stool; DI, diarrhea; HS, homosexual; - , negative; NP, nonpathogenic; ND, not done
To e v a l u a t e t h e s e n s i t i v i t y o f t h e P C R a n a l y s i s , D N A from the SAW408 strain was diluted and then amplified f o r 30 cycles. T h e r e s u l t s o f e l e c t r o p h o r e s i s o f o n e - t e n t h o f t h e a m p l i f i e d p r o d u c t s a r e i l l u s t r a t e d i n Fig. 2. W h e n t e m p l a t e D N A e q u i v a l e n t t o > 100 t r o p h o z o i t e s w a s amplified, a distinct band was observed by ethidium bromide staining. A faint band was visible even when genom i c D N A e q u i v a l e n t t o five cells w a s u s e d a s a t e m p l a t e . Such high sensitivity was on a par with that previously r e p o r t e d b y T a n n i c h a n d B u r c h a r d (1991). A l t h o u g h rela t i v e l y l a r g e n u m b e r s o f o r g a n i s m s a r e r e q u i r e d f o r zy-
modeme analysis, smaller numbers are sufficient for PCR analysis and IFA testing with mAbs. S a r g e a u n t (1988) c l a s s i f i e d 22 d i f f e r e n t t y p e s o f z y m o demes on the basis of the electrophoretic patterns of 4 isoenzymes. A more recent study suggests that the a p p e a r a n c e o f so m a n y t y p e s o f z y m o d e m e s d e p e n d e d on the culture conditions used (Blanc and Sargeaunt 1991). H o w e v e r , P C R a n a l y s i s u s i n g E. histolytica-specific p r i m e r s c o u l d m i n i m i z e t h e i n f l u e n c e o f c u l t u r e c o n d i t i o n s a n d / o r o t h e r p a r a s i t i c a m e b a e ( T a c h i b a n a e t al. 1991a). Since the possibility o f c o n v e r s i o n f r o m n o n -
436
M
1
2
3
4
5
6
7
531 bp
Fig. 2. Sensitivity of PCR amplification of genomic DNA from Entamoeba histolytica trophozoites. The DNA extracted from the SAW408 strain was serially diluted and amplified for 30 cycles using pl plus p4 primers. One-tenth of the amplified products was subjected to electrophoresis in 3% NuSieve-1% agarose gel containing ethidium bromide. The number of trophozoites equivalent to the template DNA used as shown in lanes 1-7; 0, 1, 5, 10, 10 2, 10 3, and 104 organisms, respectively; M, HincII-cleaved 9X174. The arrow indicates the position and size of PCR products p a t h o g e n i c i t y to p a t h o g e n i c i t y (or the reverse) c a n n o t be c o m p l e t e l y r u l e d o u t ( M i r e l m a n 1987; A n d r e w s et al. 1990), the g e n o t y p i c i d e n t i f i c a t i o n o f E. histolytica isolates using P C R is r e c o m m e n d e d for a sensitive a n d a c c u r a t e diagnosis.
References Andrews BJ, Mentzoni L, Bjorvatn B (1990) Zymodeme conversion of isolates of Entamoeba histolytica. Trans R Soc Trop Med Hyg 84:63-65 Blanc D, Sargeaunt PG (1991) Entamoeba histolytica zymodemes: exhibition of 7 and 6 bands only of glucose phosphate isomerase and phosphoglucomutase may be influenced by starch, content in the medium. Exp Parasitol 72:87-90 Bracha R, Diamond LS, Ackers JP, Burchard GD, Mirelman D (1990) Differentiation of clinical isolates of Entamoeba histolytica by using specific DNA probes. J Clin Microbiol 28 : 680-684 Burch DJ, Li E, Reed S, Jackson TFHG, Stanley SL Jr (1991) Isolation of a strain-specific Entamoeba histolytica cDNA clone. J Clin Microbiol 29:696-701 Edman U, Meraz MA, Rausser S, Agabian N, Meza I (1990) Char= acterization of an immunodominant variable surface antigen from pathogenic and nonpathogenic Entamoeba histolytiea. J Exp Med 172: 879-888 Garfinkel LI, Giladi M, Huber M, Gitler C, Mirelman D, Revel M, Rozenblatt S (1989) DNA probes specific for Entamoeba histolytica possessing pathogenic and nonpathogenic zymodemes. Infect Immun 57:926-931 Gongalves JF, Tanabe M, Medeiros FPM, Gongalves F J, Aca IS, Motta SRN, Tateno S, Takeuchi T (1990) Parasitological and serological studies on amoebiasis and other intestinal parasitic infections in the rural sector around Recife, northeast Brazil. Rev Inst Med Trop Sao Paulo 32:428-435
Mirelman D (1987) Effect of culture conditions and bacterial associates on the zymodemes of Entamoeba histolytiea. Parasitol Today 3 : 37-40 Nozaki T, Aca IS, Okuzawa E, Magalhges M, Tateno S, Takeuchi T (1990) Zymodemes of Entamoeba histolytica isolated in the Amazon and the north-east of Brazil. Trans R Soc Trop Med Hyg 84:387 388 Okazaki M, Miranda P, Neto J, Diegues V, Alves J, Cauas M, Tanabe M, Kobayashi S, Kaneko N, Nagakura K, Kobayashi M, Motta S, Tateno S, Takeuchi T (1988) Parasitological and serological studies on amoebiasis and other intestinal parasitic infection in Recife and its suburban area, northeast Brazil. Rev Inst Med Trop Sao Paulo 30:313 321 Robinson GL (1968) The laboratory diagnosis of human parasitic amoebae. Trans R Soc Trop Med Hyg 62:285-294 Saiki RK, Gelfand DH, Stoffel S, Scharf SJ, Higuchi R, Horn GT, Mullis KB, Erlich HA (1988) Primer-directed enzyme amplification of DNA with a thermostable DNA polymerase. Science 239:487-491 Sargeaunt PG (1988) Zymodemes of Entamoeba histolytiea. In: Ravdin JI (ed) Amebiasis: human infection by Entamoeba histolytiea. John Wiley & Sons, New York, pp 370-387 Sargeaunt PG, Baveja UK, Nanda R, Anand BS (1984) Influence of geographical factors in the distribution of pathogenic zymodemes of Entamoeba histolytica: identification of zymodeme XIV in India. Trans R Soc Trop Med Hyg 78:96-101 Tachibana H, Kobayashi S, Kato Y, Nagakura K, Kaneda Y, Takeuchi T (1990) Identification of a pathogenic isolate-specific 30,000-M, antigen of Entamoeba histolytica by using a monoclonal antibody. Infect Immun 58:955-960 Tachibana H, Ihara S, Kobayashi S, Kaneda Y, Takeuchi T, Watanabe Y (1991a) Differences in genomic DNA sequences between pathogenic and nonpathogenic isolates of Entamoeba histolytiea identified by polymerase chain reaction. J Clin Microbiol 29 : 2234-2239 Tachibana H, Kobayashi S, Nagakura K, Kaneda Y, Takeuchi T (1991 b) Reactivity of monoclonal antibodies to species-specific antigens of Entamoeba histolytiea. J Protozool 38:329-334 Takeuchi T, Kobayashi S (1983) Serological diagnosis of amoebiasis. Immunoadvance 12:27-30 Tanabe M, Okazaki M, Okazaki M, Kobayashi S, Kaneko N, Sekiguchi T, Tateno S, Motta SRN, Takeuchi T (1990) Serological studies on schistosomiasis mansoni in the northeast of Brazil (I). Rev Inst Med Trop Sao Paulo 32:121-131 Tannich E, Burchard GD (1991) Differentiation of pathogenic from nonpathogenic Entamoeba histolytica by restriction fragment analysis of a single gene amplified in vitro. J Clin Microbiol 29:250-255 Tannich E, Horstmann RD, Knobloch J, Arnold HH (1989) Genomic DNA differences between pathogenic and nonpathogenic Entamoeba histolytica. Proc Natl Acad Sci USA 86:5118-5122 Tannich E, Scholze H, Nickel R, Horstmann RD (1991) Homologous cysteine proteinases of pathogenic and nonpathogenic Entamoeba histolytiea. J Biol Chem 266 : 47984803 Walsh JA (1988) Prevalence of Entamoeba histolytica infection. In: Ravdin JI (ed) Amebiasis: human infection by Entamoeba histolytica. John Wiley & Sons, New York, pp 93-105
