RESEARCHNOTE

DETECTION OF PATHOGENIC JWTAMOEBA HTSTOLYTICA DNA IN LIVER ABSCESS FLUID BY POLYMERASE CHAIN REACTION H. TACHIBANA,*~

S. KOBAYASHI,~

E. OKUZAWA$ and G. MASUDA~

*Department of Infectious Diseases, Tokai University School of Medicine, Bohseidai, Isehara, Kanagawa 259-l 1, Japan IDepartment of Tropical Medicine and Parasitology, School of Medicine, Keio University, Shinanomachi, Shinjuku-ku, Tokyo 160, Japan §Depar~ent of Infectious Diseases, Tokyo Metro~litan Komagome Hospital, Hongo, Bunkyo-ku, Tokyo 113, Japan (Received 24 Jtdy 1992; accepted 4 September 1992)

Abstract-TACHIBANAH., KOBAYASHI S., OKUZAWA E. and MASUDA G. 1992. Detection of pathogenic Entamoeba hisrolytica DNA in liver abscess fluid by polymerase chain reaction. International Journalfor Parasitology 22: 1193-l 196. A sensitive method for detection of pathogenic Eniamoeba histo~y~ic~DNA in drained fluids from liver abscess patients, using the polymerase chain reaction (PCR), has been developed. The PCR employs oligonucieotide primers specific for the gene encoding the 30 kDa molecule of pathogenic E. hiszolytica. Liver abscess fluids (19 samples), from 14 patients with a presumptive amebic liver abscess, were examined microscopically and by the PCR method. Only two of the 19 samples were positive microscopically, whereas all 19 samples tested positive by PCR. This technique can be used to confirm the diagnosis of amebic liver abscess. INDEX KEY WORDS: Entamoeba hj~ru~~t~cQ; DNA; amebic liver abscess; polymerase chain reaction.

INTESTINALinfection with pathogenic strains of E. often leads to the development of extraintestinal disease. Liver abscess is the most common non-enteric complication of amebic infection. Since an amebic liver abscess can be lethal if adequate therapy is delayed, early diagnosis is essential. Although the most definitive method for diagnosing amebic liver abscess is to demonstrate the organisms in abscess fluid, there are limitations to the direct detection of amebae by microscopy. At present, therefore, diagnosis relies mainly on serological methods. However, in some acute cases antibody to E. histolytica cannot be detected early (Katzenstein, Rickerson & Braude, 1982), and in some individuals a positive serology may persist for a long time after recovery (Mahajan, Dutta, Chitkara, Agarwal & ~hhuttani, 1974). Recently, we reported the cloning and sequencing of the gene coding the 30 kDa molecule of E. histolytica, which can be identified by a monoclonal antibody specific to pathogenic strains (Tachibana, Kobayashi, Kato, Nagakura, Kaneda & Takeuchi, 1990; Tachibana, Ihara, Kobayashi, Kaneda, Takeuchi & Watanabe, 199 1). Direct sequencing of genomic DNA amplified by the polymerase chain reaction (PCR) has histolytica

t To whom all correspondence should be addressed.

revealed a 5% difference in the nucleotide sequences between pathogenic and non-pathogenic strains. It was shown that the combined use of PCR and restriction enzyme digestion, or PCR alone, is a useful strategy for distinguishing pathogenic isolates of E. histolytica from non-pathogenic isolates and other non-pathogenic amebae (Tachibana, Kobayashi, Takekoshi & Ihara, 1991; Tachibana, Kobayashi, Paz, Aca, Tateno & Ihara, 1992). In this paper we report the application of the PCR technique for the detection of DNA of pathogenic E. histolytica organisms in liver abscess fluids. Nineteen abscess fluids were obtained from 14 patients with amebic liver abscesses before and/or after the start of metronidazole therapy. The presumptive diagnosis of amebic liver abscess was based on clinical symptoms, positive serology, ultrasound examination, and prompt response to treatment with metronidazole. Serologic examination was by the geldiffusion precipitin test and enzyme-linked immunosorbent assay (ELISA), as previously described (Takeuchi & Kobayashi, 1983; Takeuchi, Matsuda, Okuzawa, Nozaki, Kobayashi & Tanaka, 1988). The presence of trophozoites in liver abscess fluids was determined by microscopy immediately and after cultivation of sample aliquots in Robinson’s medium. Positive cultures were subjected to zymodeme analysis

1193

H.

1194 TABLE l-DETECTIONOF

TACHIBANA~~~.

Entamoeb~his~oly~ica DNA~~twea (PCR)

ABSCESSFLUIDSBY THE POLYMERASE CHAIN REACTION

Patient No.

1 2 3

4 5 6 7 8 9 10 11 12 13 14

Metronidazole: days after start of treatment

0 4 0 15 0 4 13 0 5 0 0 0 2 3 4 4 4 5 6

Microscopic Serology detection of parasites (zymodeme)* + + _ _. _ -

_ -

(Z-II)

+

(Z-XI)

+ f

+ + + + + + + + + + + +

PCR

+ + + + + + + + + + + + + + + + + + +

*Zymodemes were determined after cultivation

according to the procedure of Sargeaunt, Nanda & Anand (1984). Five aspirates

Baveja,

of liver abscesses from patients free of symptoms of amebic infection and with a negative serology were used as controls. A 100 ~1 aliquot of each liver abscess fluid was diluted with an equal volume of TE buffer [lo tIIMTri-HCI (pH 7.5), 1 mM-EDTA], mixed with 200 ~1 of lysis buffer containing 200 mM-NaCI, 20 mM-EDTA, 20 mM-Tris-HCt (pH 8.0), 4% (w/v) sodium dodecyl sulfate, and 1 mg ml-’ proteinase k, then incubated for I h at 60°C. DNA was extracted twice with an equal volume of phenol/~hlorofo~/isoamyl alcohol (25:24:1). The DNA was ethanol-precipitated with sodium acetate at - 80°C for 30 min, then pelleted by centrifugation at 10,000 g for 10 min at 4°C. The pellets were washed in 70% ethanol, centrifuged, dried and resuspended in 100 ,uI of TE buffer. An aliquot of extracted DNA in TE buffer was subjected to 45 cycles of amplification in a 50 ~1 volume containing I U of 7hy DNA polymerase (Wako, Osaka, Japan), 1 ,UM of each of the two primers, 200 PM of each of the four deoxynucleotides, 10 miw-Tris-HCI (pH 8.8), 50 mM-KCi, 1.5 mM-MgCI,, and 0.1% Triton X-100. The oligonuc~eotide primers, pl 1 (5’-GGAGGAGTAGGAAAG~GAC-3’) and pi2 (5’-~C~GCAA~CCTGC~CGA-3’), were

used for PCR amplification. These primers amplify a 100 bp sequence of the gene coding the 30 kDa molecule of pathogenic strains of E. hist~iy~~ca (Tachibana, Kobayashi, Takekoshi & Ihara, 1991). The mixtures were overlaid with one drop of mineral oil. After an initial denaturation step at 94°C for 2 min, each amplification cycle was performed as follows: 1 min for denaturation at 94”C, 1.5 min for annealing at 59”C, and 1.5 min for primer extension at 72°C. Ten microliters of amplified sample were electrophoresed in 2% agarose gel containing 0.5 ,Q ml”’ ethidium bromide and the gel was photographed under U.V. light. Samples containing a band of the expected size were subjected to EcoT221 cleavage according to the manufacturer’s recommendation (Toyobo, Osaka, Japan) and analyzed by gel electrophoresis. When liver abscess fluids were examined directly and after cultivation for the presence of trophozoites by microscopy, only two of seven samples from patients who had not received metronidazole were positive (Table 1). No organisms were detected in specimens collected 2-15 days after the start of chemotherapy. In preliminary PCR studies, the optimal concentration of template DNA for PCR amplification was determined by the use of increasing amounts of extracted DNA from the above E. histo&t&-positive two samples. When 5 or more ~1 of extracted DNA solution were used as a template, a reduction in the intensity of the 100 bp band was observed in both samples (data not shown). This reduction may be due to the large amount of host DNA in the samples. When 0.5-2 ~1 were used as a template, optimum intensity of the 100 bp band was achieved. Therefore, 2 ~1 of DNA solution (equivalent to 2 ~11of abscess fluid) were routinely used in all the PCR amplification tests. In this manner, the expected 100 bp products were amplified even in the 17 samples which were microscopically negative (Fig. 1. lanes l10, Table 1). In contrast, no products were amplified in any of the five control samples collected from patients with non-amebic liver abscesses (Fig. 1, lanes 11-i 3). Genetic differences between pathogenic and nonpathogenic strains of E. histoiytica have been reported recently (Tannich, Horstmann, Knobloch & Arnold, 1989; Clark & Diamond, 1991; Tachibana, Ihara, Kobayashi, Kaneda, Takeuchi & Watanabe, 1991; Tannich, Scholze, Nickel & Horstmann, 1991). Since the primers used in the present study were specific for the pathogenic isolates of E. histoiytica, it seems very likely that the amplified PCR products were derived from pathogenic E. hjszol~~~cu (Tachibana, Kobayashi, Takekoshi & Ihara, 1991). For further confirmation,‘these products were subjected to digestion with EcoT221 because a comparable sequence in

Research Note

1195

FIG. 1. Agarose gel separation of polymerase chain reaction products amplified from pathogenic Entumoeba histolytica in liver abscess fluids using primers derived from the 30 kDa molecule sequence. Patient number and (days) after the start of the treatment, lanes l-10: lane 1, patient 1 (0); lane 2, patient 1 (4); lane 3, patient 2 (0); lane 4, patient 2 (15); lane 5, patient 6 (0); lane 6, patient 8 (2); lane 7, patient 10 (4); lane 8, patient 11 (4); lane 9, patient 13 (5); lane 10, patient 14 (6). Lanes 11-13 (controls), patients with non-amebic liver abscesses; lane 14 (control), PCR performed without template DNA; lane M, DNA size marker (100 bp ladder).

non-pathogenic strains does not contain the cleavage site of EcoT221 (Tachibana, Ihara, Kobayashi, Kaneda, Takeuchi & Watanabe, 1991). The results showed that the 100 bp amplified products from the 19 abscess samples were cut into two segments as expected, indicating that the E. histolytica DNA present in the abscess fluids apparently was derived from a pathogenic strain in every case (data not shown). Likewise, isoenzyme analysis of the two isolated parasites showed pathogenic zymodeme patterns, Z-II and Z-XI (Table 1). These observations support the concept that E. histofytica consists of two different types, only one of which is an invasive pathogen. As alternatives to microscopy for the difficult diagnosis of amebic liver abscess, the detection of amebic antigens by counterimmunoelectrophoresis and ELISA has been used (Mahajan & Ganguly, 1980; Bhave, Wagle & Joshi, 1985). The sensitivity of these methods, compared to a presumptive diagnosis of amebic liver abscess (based on clinical criteria, plus aspiration of bacteriologically sterile abscess fluid and complete cure following anti-amebic chemotherapy), was scored as 92 and 96%, respectively. The present study indicates that the detection of E. histolytica DNA by PCR is even more sensitive although the number of samples examined were limited. Advances in modern imaging techniques have promoted the use of guided percutaneous aspiration and drainage. Although the use of such procedures in cases of amebic liver abscess is still controversial, it was shown that percutaneous drainage using ultrasound guidance followed by metronidazole administration resulted in faster resolution, fewer relapses, and less

residual hepatic scarring than with chemotherapy alone (Filice, Di Perri, Strosselli, Brunetti, Dughetti, Van Thiel & Scotti-Foglieni, 1992). As shown in this study, by using PCR amplification, pathogenic E. histolytica DNA can be clearly detected in a small amount of abscess fluid. Therefore, if abscess fluid is obtainable, the PCR method as reported herein will permit a positive diagnosis even in the absence of microscopic detection of the parasites. Acknowledgements-We are grateful to Professors Y. Kaneda and T. Takeuchi for their encouragement of this study. We also thank Dr W. Stahl for reviewing the manuscript. This work was supported by a Grant-in-Aid for Scientific Research from the Ministry of Education, Science and Culture of Japan; a Grant-in- Aid for a DNA diagnosis project of the Tokai University School of Medicine; a Tokai University School of Medicine Research Aid; and by a grant from the Ohyama Health Foundation to H. T. REFERENCES BHAVEG. Ci., WAGLEN. M. & JOSHI U. M. 1985. Detection of amoebic antigen by enzyme linked immuno-sorbent assay (ELISA). Journal of Postgraduate Medicine 31: 146-149. CLARKC. G. & DIAMONDL. S. 1991. Ribosomal RNA genes of ‘pathogenic’ and ‘nonpathogenic’ Entamoeba histolytica are distinct. Molecular and Biochemical Parasitology 49: 297-302. FILICE C., DI PERRI G., STROSSELLIM., BRUNE~I E., DUGHETTIS., VAN THIELD. H. & SCOITI-FOGLIENI C. 1992. Outcome of hepatic amebic abscesses managed with three different therapeutic strategies. Digestive Diseases and Sciences 31: 24&247. KATZENSTEIND., RICKERSONV. & BRAUDE A. 1982. New concepts of amebic liver abscess derived from hepatic

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