multiplex polymerase chain reaction amplification and differentiation of ...

Am. J. Trop. Med. Hyg., 64(5, 6), 2001, pp. 293–297 Copyright 䉷 2001 by The American Society of Tropical Medicine and Hygiene

MULTIPLEX POLYMERASE CHAIN REACTION AMPLIFICATION AND DIFFERENTIATION OF ENTAMOEBA HISTOLYTICA AND ENTAMOEBA DISPAR DNA FROM STOOL SAMPLES Ń ˜ EZ, MARIÁ A. FERNA ´ NDEZ, DIAMELA TORRES-NU Ń ˜ EZ, JOSE ´ A. SILVA, IVO ´ N MONTANO, YURY O. NU JORGE L. MAESTRE, AND LUIS FONTE Institute of Tropical Medicine Pedro Kourı´, Havana, Cuba; Center for Medical and Surgical Researches, Havana, Cuba; Center for Genetic Engineering and Biotechnology, Havana, Cuba

Abstract. Due to the clinical importance of differentiating the two species of the Entamoeba histolytica/Entamoeba dispar complex, we developed a multiplex polymerase chain reaction (PCR) method that overcomes time-consuming and laborious procedures. We report here a DNA extraction protocol using non-fixed stool samples that avoid long lysis-incubation periods through the combined use of zirconium beads and a lysis-supporting buffer. We characterized 49 of 52 stool specimens from Cuban patients with amoebiosis. Among them, 36 (75.5%) were infected only with E. dispar (the nonpathogenic species), while 13 (24.5%) displayed a mixed infection with both E. dispar and E. histolytica. The multiplex PCR protocol showed a specificity of 1.00 and a sensitivity of 0.94. Furthermore, the entire procedure can be performed in one day. This approach is therefore reliable and applicable in the field for epidemiologic studies. E. histolytica/E. dispar.16 Among the arguments in favor of ELISA versus PCR are the convenience and lower prices of ELISA-based kits, especially for a routine diagnostic laboratory in endemic areas. However, as reported by Mirelman and others,16 improvements in automation and simplification of PCR procedures for clinical sampling directly from stools, as well as decreased price of equipment, reagents, and product detection systems that have been recently achieved, indicate that a comparison with the ELISA needs to be carefully reevaluated. In this report, we describe the development of a new PCRbased approach for the detection and characterization of the species of the E. histolytica/E. dispar complex. We used a multiplex PCR methodology to amplify simultaneously two species-specific DNA fragments using two pairs of specific primers combined in a single reaction mixture. This procedure therefore saves considerable time and resources. This approach was originally developed by Chamberlain and others26,27 to detect human genes, and later modified by Bej and others28 to detect gene sequences associated with different groups of bacteria in environmental samples. However, despite the economical and operational advantages provided by this approach, it has not been frequently applied in novel PCR-based strategies used in research and diagnosis. The basis behind this modified technique is the design of primers that all have a similar annealing temperature (Ta) and similar target DNA sizes, as large differences in the length of target DNAs will favor the amplification of the shortest target over the longer one.29

INTRODUCTION

Amoebic infections result either in a harmless colonization of the intestine, or in invasion and damage (amoebiosis) of other host tissues such as the liver, lung, and brain. These distinct manifestations are due to the existence of Entamoeba histolytica/Entamoeba dispar as a complex of two different, but morphologically identical species: one that is a nonpathogenic commensal in the intestine of humans (E. dispar Brumpt, 1925), and the other that is capable of inducing cell and tissue damage (E. histolytica Schaudinn, 1903). This assertion derives from extensive immunological, biochemical, and molecular biological data that indicate they have a high degree of divergence and are, in fact, two separate species.1–9 A clinical diagnosis of amoebiosis can be confirmed by microscopic identification of characteristic cysts or trophozoites in the stool. However, microscopic examination has several limitations,10,11 the most important being the inability to distinguish E. histolytica from E. dispar. In addition, multiple samples often have to be examined and the presence of cysts of different species such as Entamoeba, Iodamoeba, or Endolimax can make diagnosis difficult. The epidemiology of Entamoeba can be further studied by culturing trophozoites and determining isoenzyme patterns by gel electrophoresis. However, these techniques are laborious, expensive, and time-consuming, and are not practical for routine diagnostic laboratories.12,13 Detection of antibodies to amoeba in patient sera has been reported to indicate infection by E. histolytica.14 However, with serological testing, it may be difficult to distinguish past from present infections in individuals who emigrate from, or currently reside in, endemic areas.15 New diagnostic approaches are based on antigen detection in stool and on several molecular biology techniques.11,13, 15–25 Stool antigen assay is as sensitive and specific as isoenzyme analysis and outperforms microscopy in the detection of E. histolytica.11,13 Nevertheless, comparative results clearly indicate the advantages of the polymerase chain reaction (PCR) compared with enzyme-linked immunosorbent assay (ELISA)-based kits in the detection and characterization of

MATERIALS AND METHODS

Samples. Stool samples were collected from individuals who sought medical attention for different reasons, most frequently diarrheal diseases, at the parasitology services of two hospitals and 13 polyclinics in Cienfuegos Province in Cuba from May to September 1996. All specimens were studied by routine procedures used for microscopic examination of feces in the corresponding health centers. To confirm infection with microorganisms of the E. his-

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tolytica/E. dispar complex, each sample was frozen at ⫺20⬚C without formalin, sent to the Parasitology Laboratory of the Tropical Medicine Institute Pedro Kourı´ (IPK) in Havana (300 km away), and analyzed using the ENZYMEBA威 test (IPK, Havana, Cuba). This test is a commercial enzyme immunoassay kit produced by the IPK, and was used according to the manufacturer’s instructions, based on the procedure described by Luaces and others.30 Samples were divided into 3 groups: 1) an E. histolytica/ E. dispar-positive group with 52 individuals who tested positive by both microscopic examination and the ENZYMEBA威 test; 2) a negative control group including samples from 30 individuals found to be negative by microscopic examination and the ENZYMEBA威 test; and 3) a cross-reaction control group with 40 patients infected with other parasites, including Blastocystis hominis (5 patients), Entamoeba coli (3 patients), Giardia lamblia (5 patients), Endolimax nana (5 patients), Schistosoma mansoni (5 patients), Fasciola hepatica (5 patients), Trichuris trichiura (4 patients), Taenia saginata (2 patients), Ascaris lumbricoides (2 patients), and Necator americanus (4 patients). All patients in this group were confirmed negative for E. histolytica/E. dispar by ENZYMEBA威 test. Extraction of DNA from stool samples. All procedures were performed using sterile, disposable plastic tubes and pipette tips. DNA was extracted according to the following protocol. Feces (0.5 grams) were placed in a 1.5-ml microcentrifuge tube, washed once with 1 ml of phosphate-buffered saline solution (PBS; 0.1 M Na2HPO4, 0.1 M NaH2PO4, pH 7.5), and filtered through gauze. The feces samples must be washed with PBS before lysis of cyst and trophozoites to eliminate soluble contaminants that affect the specificity of the PCR and yield of amplification. The filtered supernatant was centrifuged at 3000 ⫻ g for 5 min, resuspended in 500 ␮l of lysis-supporting buffer (LSB; 0.1 M Tris-Cl, pH 8.0, 0.1 M EDTA, 0.1 M NaCl, 2% sodium dodecyl sulfate), and added to a 2-ml capped tube containing zirconium beads (0.1–0.15 mm, volume ⬃500 ␮l (Biospect Products, Inc., Battersville, OK) and 500 ␮l of phenol (Tris-equilibrated, pH 8.0). The mixture was vortexed for 2 min and centrifuged at 12,000 ⫻ g for 15 min. The aqueous layer was recovered, extracted with chloroform: isoamyl alcohol (24:1), and the DNA was precipitated with one volume (⬃500 ␮l) of isopropanol. The pellet was resuspended in 100 ␮l of TE buffer (10 mM Tris-Cl, pH 8.0, 10 mM EDTA). The use of LSB during the lysis step, in addition to the zirconium beads, was necessary to efficiently disrupt most of the cysts present in the feces. We compared mechanical lysis using the zirconium beads with and without LSB, and found an increased DNA yield and PCR sensitivity when LSB was used. Isopropanol was used to selectively precipitate DNA without the carbohydrates that are abundant in Entamoeba and could interfere with the amplification reactions. This protocol resulted in the isolation of DNA of sufficient quality and quantity for sensitive and accurate PCR amplification. Multiplex PCR amplification. The PCR was performed in a total volume of 50 ␮l in 0.5-ml PCR plastic tubes, using a PTC-100娂 Thermal Controller (Peltier-Effect Cycling; MJ Research, Inc., Waltham, MA). Reaction conditions were optimized to combine two unrelated reactions using a mixture of two specific primer pairs. Each primer set yielded a 96

base pair (bp) product for E. dispar (EdP1/2) and a 132 bp product for E. histolytica (EhP1/2). Primer construction was based on the reported E. histolytica P145 and E. dispar B133 sequences, which are tandemly repeated in the respective extrachromosomal circular DNAs.31–33 These sequences are species-specific and are therefore suitable for genetic characterization. The primer sequences used were EdP1: 5⬘ATGGTGAGGTTGTAGCAGAGA-3⬘, EdP2: 5⬘-CGATATTGGATACCTAGTACT-3⬘, EhP1: 5⬘-CGATTTTCCCAGTTAGAAATTA-3⬘, and EhP2: 5⬘-CAAAATGGTCGTCGTCTAGGC-3⬘. The reaction mixture contained 250 ␮M each dNTPs, 40 pmoles of each primer, 1.5 mM MgCl2, 1.25 units of Taq polymerase, 1⫻ Taq buffer, and 10 ␮l of the extracted DNA solution. Amplification of DNA was carried out for 30 cycles with denaturation at 94⬚C for 30 sec, primer annealing at 55⬚C for 30 sec, and extension at 72⬚C for 30 sec. Four controls were included in all experiments: 1) all reagents except DNA template, 2) control DNA from E. histolytica, 3) control DNA from E. dispar, and 4) a mixture of control DNA from E. histolytica and E. dispar. Amplified products were visualized with ethidium bromide after electrophoresis on 10% acrylamide gels. Acrylamide gels were used to ensure proper differentiation of the amplified products, which differ in length by just 36 nucleotides. Control DNA of E. histolytica and E. dispar. DNA from the HM1-IMSS strain was used as the reference for E. histolytica, and DNA isolated from a cultured sample from an asymptomatic infected patient and characterized by isoenzyme analysis was used as the reference for E. dispar. Determination of the multiplex PCR detection limits. To define the minimum detectable amount of E. histolytica and E. dispar, variable quantities (105, 104, 103, and 102 parasites) of both control strain were mixed with 0.5 grams of feces from a sample of the negative control group, and DNA was extracted and assayed by the PCR using the same protocols described earlier. RESULTS Primer specificity. Amplification was specific for each primer pair (Figure 1, lanes 2–5). The E. histolytica primers (EhP1/2) amplified DNA from the HM1-IMSS strain but not from E. dispar whereas the E. dispar primers (EdP1/2) amplified DNA from E. dispar but not from HM1-IMSS. When parasites from both control strains were mixed and specific DNAs were amplified using a mixture of the two primer pairs, the two 96 and 132 bp fragments were visualized after electrophoresis and staining with ethidium bromide. No interference was noted between the two amplification systems (Figure 1, lanes 6–9). Detection limit. As shown in Figure 1, both E. histolytica and E. dispar DNA were detected by the multiplex PCR, even at the minimum parasite concentration tested (100 parasites/0.5 grams of feces). This indicates that up to 10⫺1 pg of DNA could be detected by this procedure as only 10 ␮l DNA (100 parasite/100 ␮l of TE) was used for amplification. Multiplex PCR specificity and sensitivity. As shown in Table 1, no products were detected when samples from the negative control group and the cross-reaction control group were tested by the multiplex PCR. This represents a maximum specificity (1.00) and no collateral cross-reactions. Re-

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FIGURE 1. Results of the polymerase chain reaction (PCR) amplifications. Lane 1, molecular weight marker (pBR322 ⫹ Hpa II); lanes 2 and 3, single PCR with HM1:IMSS DNA using primers EdP1/2 and EhP1/2, respectively; lanes 4 and 5, single PCR with Entaoeba dispar DNA using primers EdP1/2 and EhP1/2, respectively; lanes 6–9, multiplex PCR for detection limit determination (DNA from mixtures of 105, 104, 103, and 102 parasites of each species in 0.5 grams of feces, respectively); lane 10, negative control. Molecular weight markers (in bp) are 527, 404, 242, 238, 217, 201, 190, 180, 160, 147, 123, 110, 90, 76, and 67.

sults of the multiplex PCR with samples from infected individuals showed a sensitivity of 0.94 and indicated circulation of both E. histolytica and E. dispar in the Province of Cienfuegos, Cuba. DISCUSSION We described a new PCR-based approach for accurate detection and differentiation of the two species that compose the E. histolytica/E. dispar complex, by extracting DNA directly from stool samples without prior cultivation. The protocols are fast and simple, no laborious hybridizations or use of radionucleotides are required, and the entire procedure can be performed in one day. In addition, it is not necessary to perform two amplification reactions separately with the same sample as a single reaction can determine if patients were infected with E. histolytica, E. dispar, or both species. This approach is relevant for epidemiological studies, where many samples are examined and diagnosis can be expensive and laborious. We characterized 49 of 52 individuals infected with microorganisms of the E. histolytica/E. dispar complex. Microscopic detection of parasites from the E. histolytica/E.

dispar complex was confirmed using the ENZYMEBA威 test. Because this test requires that feces samples are stored frozen without formalin (which is used to conserve cysts in samples for characterization by PCR), this could explain the 3 negative results. Of the 49 PCR-positive samples, 36 (75.5%) were infected only with the nonpathogenic E. dispar, and 13 (24.5%) showed mixed infections with the two species. It is interesting that no single E. histolytica infections were found in our study. Other investigators have also found that infection with E. dispar is more common than infection with E. histolytica.17,34–38 Evidence of the inverse proportion has been reported by Acuna-Soto and others,21 who targeted the same specific and tandemly repeated DNA sequences described in the current study and found E. histolytica as the predominant population. Similarly, the occurrence of mixed infections with both E. histolytica and E. dispar has been reported,11,15,17,21,38 as well as association among these two species and Endolimax nana and Entamoeba coli,21,39,40 suggesting that there may be common mechanisms of transmission or specific susceptibility. It can be inferred from previous reports that differentiation by culture/isoenzyme analysis or antigen detection kits could overlook mixed infections with E. histolytica and E. dispar, since they were detected only after a PCR was used directly on the same stool specimens previously assayed.11,15 Regarding antigen-detection methods, a small number of E. dispar organisms present among a large population of E. histolytica would be difficult to detect using an E. histolytica-specific monoclonal antibody (MAb) and an MAb cross-reactive with E. histolytica and E. dispar.41 In such cases, only the use of a PCR or E. histolytica- and E. dispar-specific MAbs would facilitate differentiation. These observations could explain why mixed infections with E. histolytica and E. dispar were not reported before the development of more specific and sensitive molecular techniques. There have been several reports regarding the inability to detect mixed E. histolytica/E. dispar infections.34,37,38,41,42 All of these studies, except that of Rivera and others,37 reported prior cultivation of the clinical specimens before assaying with techniques involving antigen detection, zymodemes, or a PCR. As suggested by Valdez and others,42 it is possible that cultures of cysts obtained from human samples undergo some kind of selection with regard to the species of Entamoeba isolated. Interestingly, Rivera and others,37 found a clear association between E. histolytica/E. dispar and Entamoeba coli in terms of coinfection, although no E. histo-

TABLE 1 Multiplex polymerase chain reaction (PCR) for Entamoeba histolytica (Eh)/E. dispar (Ed), using primers EdP1, EdP2, EhP1, and EhP2 in different groups of fecal samples ⫹PCR (%)* Group

Negative control Cross-reaction control group Individuals with confirmed E. histolytica/E. dispar infection

n

Only Eh†

30 40 52

Only Ed†

Mixed†

0 (0) 0 (0) 49 (94.2) 0 (0)

36 (75.5)

⫺PCR (%)

30 (100) 40 (100) 3 (5.8) 13 (24.5)

* When ⫹ PCR results were 0 (0) for any group, no additional calculation was necessary for discrimination among only Eh, only Ed, or mixed infections. † This percentage was recalculated taking as 100% the actual total number of samples positive for Eh or Ed as determined by our multiplex PCR assay.

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lytica/E. dispar mixed infections were detected. They did not discount the possibility of a competitive phenomenon in vivo between E. histolytica and E. dispar, but referred to an in vitro study43 showing that only a minuscule amount of E. histolytica can ultimately outgrow E. dispar in culture in a given period of time. In contrast, Gatti and others44 reported that some pathogenic amoebic zymodemes outgrow others. These findings suggest that an adequate animal model of amoebiosis needs to be developed as a prerequisite to clarify this phenomenon, as suggested by Rivera and others.37 Our results provide important data for the Public Health Care System in Cuba because this is the first time the species of the E. histolytica/E. dispar complex circulating in this country have been characterized at the DNA level. Mendiola and others,45 using biochemical characterization by zymodeme patterns of amoebas cultured from stool specimens, reported that pathogenic E. histolytica was not present in Cuban patients. Our observations indicate a possible recent introduction of E. histolytica into Cuba, and as E. dispar seems to be endemic, it could explain the absence of single E. histolytica infections. In conclusion, our data indicates a need to address the emerging problem of amoebiosis in our country. Acknowledgments: We thank Dr. Humberto Marıń for making available the stool specimens used in this study, Dr. Judith Mendiola for providing the E. dispar control strain, and Dr. Esther Orozco for providing the HM1:IMSS control strain. Authors’ addresses: Yury O. Nu´ n˜ ez*, Diamela Torres-Nu´ n˜ ez†, Ivo´ n Montano, Jorge L. Maestre, and Luis Fonte, Departamento de Parasitologıá y Departamento de Microbiologıá, Instituto de Medicina Tropical Pedro Kourı´, Apartado Postal 601, Marianao 13, Ciudad de la Habana, Cuba. Marıá A. Ferna´ ndez, Departamento de Microbiologıá, Centro de Investigaciones Me´ dico Quiru´ rgicas, Calle 214 e/ 13 y 17, Reparto Siboney, Ciudad de la Habana, Cuba. Jose´ A. Silva, Departamento de Sıńtesis de Oligonucleo´ tidos, Centro de Ingenierıá Gene´ tica y Biotecnologıá, Apartado Postal 6162, Avenue 31 e/158 y 190, Cubanacan, Ciudad de la Habana, Cuba. *Y.O.N.’s current address: Molecular Biology Program, RRL-937 Box 86, Memorial Sloan-Kettering Cancer Center, 1275 York Ave., New York City, NY 10021, e-mail: [email protected]. †D.T-N.’s current address: Department of Research and Development, Amersham Pharmacia Biotech, Inc., 800 Centennial Avenue, PO Box 1327, Piscataway, NJ 08855–1327, e-mail: [email protected].

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