Journal of Helminthology (2015) 89, 769–773 q Cambridge University Press 2014
doi:10.1017/S0022149X14000595
Polymerase chain reaction for the amplification of the 121-bp repetitive sequence of Schistosoma mansoni: a highly sensitive potential diagnostic tool for areas of low endemicity E. Ferrer1,2*, F. Pe´rez1, I. Bello1, A. Bolı´var1, M. Lares1, A. Osorio3, L. Leo´n3, M. Amarista4 and R.N. Incani5 1
Instituto de Investigaciones Biome´dicas ‘Dr. Francisco J. Triana Alonso’ (BIOMED), Facultad de Ciencias de la Salud, Universidad de Carabobo Sede Aragua, Maracay, Venezuela: 2Departamento de Parasitologı´a, Facultad de Ciencias de la Salud, Universidad de Carabobo Sede Aragua, Maracay, Venezuela: 3Programa Nacional de Prevencio´n y Control de Parasitosis Intestinales y Esquistosomosis, Direccio´n General de Salud Ambiental, Ministerio del Poder Popular Para La Salud, Maracay, Venezuela: 4Centro de Estudios de Enfermedades Ende´micas y Salud Ambiental (CEEESA), Instituto de Altos Estudios ‘Dr. Arnoldo Gabaldo´n’, Ministerio del Poder Popular Para La Salud, Maracay, Venezuela: 5Laboratorio de Investigaciones en Bilharzia, Departamento de Parasitologı´a, Facultad de Ciencias de la Salud, Sede Carabobo, Universidad de Carabobo, Valencia, Venezuela (Received 23 April 2014; Accepted 11 July 2014; First Published Online 20 August 2014) Abstract Schistosomiasis is a disease caused by parasitic flatworms of the genus Schistosoma, whose diagnosis has limitations, such as the low sensitivity and specificity of parasitological and immunological methods, respectively. In the present study an alternative molecular technique requiring previous standardization was carried out using the polymerase chain reaction (PCR) for the amplification of a 121-bp highly repetitive sequence for Schistosoma mansoni. DNA was extracted from eggs of S. mansoni by salting out. Different conditions were standardized for the PCR technique, including the concentration of reagents and the DNA template, annealing temperature and number of cycles, followed by the determination of the analytical sensitivity and specificity of the technique. Furthermore, the standardized PCR technique was employed in DNA extracted, using Chelexw100, from samples of sera of patients with an immunodiagnosis of schistosomiasis. The optimal conditions for the PCR were 2.5 mM MgCl2, 150 mM deoxynucleoside triphosphates (dNTPs), 0.4 mM primers, 0.75 U DNA polymerase, using 35 cycles and an annealing temperature of 638C.
*Fax: þ58 2435333 E-mail: [email protected]
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The analytical sensitivity of the PCR was 10 attograms of DNA and the specificity was 100%. The DNA sequence was successfully detected in the sera of two patients, demonstrating schistosomiasis transmission, although low, in the community studied. The standardized PCR technique, using smaller amounts of reagents than in the original protocol, is highly sensitive and specific for the detection of DNA from S. mansoni and could be an important tool for diagnosis in areas of low endemicity.
Introduction
Materials and methods
Schistosomiasis is a disease caused by parasitic flatworms of the genus Schistosoma. The disease is a major public health problem, affecting more than 240 million people in developing countries. Schistosoma mansoni is endemic in 54 countries in South America, the Caribbean, Africa and the eastern Mediterranean region (WHO, 2014). Currently schistosomiasis is endemic in the Americas. It is a predominantly rural parasitosis, although it may be suburban and even urban, depending on the existence of the conditions for transmission. Socio-economic factors influence the transmission of schistosomiasis, being prevalent in tropical and subtropical areas, in poor communities without potable water and adequate sanitation (Barreto, 1991; WHO, 2014). The actual epidemiological situation of schistosomiasis in Venezuela is of low transmission (Alarco´n de Noya et al., 2007), which makes the parasitological diagnosis by standard coprology difficult. Serological diagnosis is carried out in Venezuela using the cumbersome goldstandard circumoval precipitin test (COPT), in addition to two other tests, the alkaline phosphatase immunoassay (Pujol et al., 1989; Pujol & Cesari, 1990) and the Western blot (Cesari et al., 2005) – both tests of experimental use, requiring a reliable validation test, since the prevalence value for both tests is much higher than that obtained by COPT. Molecular diagnosis using polymerase chain reaction (PCR) appears to be an alternative. The PCR systems described are sensitive and have the potential to detect parasite DNA in snails, monitor cercariae in water bodies (Hamburger et al., 1991, 1998a, b; Abath et al., 2006) and diagnose human infection (Pontes et al., 2002, 2003; Abath et al., 2006). More extensive validation studies in endemic areas are crucial to investigate the suitability of this molecular tool for the diagnosis of infected humans. Standardization of a protocol for PCR amplification of the 121-bp highly repetitive sequence of S. mansoni (Pontes et al., 2002) could provide a technique that allows rapid, accurate, sensitive and specific diagnosis, which could be used for both clinical diagnosis and epidemiological studies, being able to distinguish active from non-active foci, a fact not easily attainable using immunological methods since antibodies may persist over time, while the presence of parasite DNA depends on an active infection. In addition, making the proper diagnosis of active outbreaks can provide benefits for the treatment of infected humans, and surveillance of infection in vectors and waters will be more reliable for decisions to be taken by a Schistosomiasis Control Programme.
Collection of samples In order to standardize protocols of the PCR for the amplification of a 121-bp highly repeated sequence of S. mansoni (Pontes et al., 2002), egg samples of the Brazilian BH strain of S. mansoni obtained from experimentally infected golden hamsters were used. In addition, DNA samples of other parasites, such as Fasciola hepatica, Taenia solium, Taenia saginata, Toxocara canis, Trypanosoma cruzi, Leishmania chagasi and Toxoplasma gondii, and human DNA were used to determine the specificity of PCR. In addition, serum samples from 86 humans with immunological diagnosis of schistosomiasis from an ancient endemic focus of this disease (Barrio Nuevo, Tiara, Aragua State) were analysed. The patients were treated 1 year previously with praziquantel, and evaluated a year after treatment. The results showed that all patients were negative by coprological diagnosis (Kato – Katz) and positive by immunological diagnosis (Western blot). These serum samples were used to evaluate if there was active transmission at the time of evaluation, or if the immunological results were due to past infections. Molecular analysis Conventional methods for the extraction of DNA, such as extraction by salting out, and the use of Chelexw 100 resin (BioRad Laboratories, Redmond, Washington, USA) were used. DNA extraction by the salting-out technique was performed according to the original protocol described by Sambrook & Russell (2001), but increasing the time of incubation with proteinase K to 4 h and the centrifugation time after incubating with proteinase K to 30 min, to match the type of sample (parasite eggs). DNA extraction from serum samples from 86 patients with immunological diagnosis of schistosomiasis, using resin Chelexw 100, was done following the instructions of the manufacturer. The PCR for the amplification of the 121-bp highly repeated sequence of S. mansoni (Pontes et al., 2002) was carried out using DNA samples extracted from parasite eggs obtained from the liver of experimentally infected golden hamsters. The amplification reactions were carried out according to the protocol described by Pontes et al., 2002), using the forward 50 -GATCTGAATCCGACCAACCG-30 and reverse 50 -ATATTAACGCCCACGCTCTC-30 primers and varying the concentrations of
771
Molecular diagnosis of schistosomiasis Table 1. The concentration and quantity of reagents, including conditions tested, for the PCR amplification of the 121-bp repetitive sequence of S. mansoni. Reagent/condition
Quantity/concentration/condition tested
DNA
1 mg, 100 ng, 10 ng, 1 ng, 100 pg, 10 pg, 1 pg, 100 fg, 10 fg, 1 fg, 100 ag, 10 ag, 1 ag, 0.1 ag 1 mM , 1.5 mM , 2 mM , 2.5 mM 100 mM , 150 mM , 200 mM , 250 mM 0.2, 0.4, 0.6, 0.8 mM 0.5 U, 0.75 U, 1.0 U, 1.25 U 608C, 638C 30, 35
Magnesium chloride dNTPs Primers Taq DNA polymerase Annealing temperature Number of cycles
reagents, such as deoxynucleoside triphosphates (dNTPs), primers, polymerase and magnesium chloride, and conditions of temperatures and number of cycles (table 1), to verify the conditions for the best amplification. PCR products were visualized in 2% agarose gels and compared with molecular size markers. All tested conditions for PCR were compared, and they were selected as optimal, where the expected bands were clear and with the absence of non-specific products or smears, observed through photographic images of agarose gels. The analytical sensitivity was determined using different quantities of DNA, 1 mg to 0.1 attogram (at) (table 1). The specificity was also determined using DNA samples from other parasites, such as F. hepatica, T. solium, T. saginata, T. canis, T.cruzi, L. chagasi and T. gondii, and human DNA. The previously standardized PCR for the amplification of the 121-bp highly repeated sequence of S. mansoni (Pontes et al., 2002) was performed using DNA samples extracted from serum samples from 86 patients with immunological diagnosis of schistosomiasis.
Results and discussion In Venezuela, transmission of S. mansoni can be characterized as hypoendemic and infected patients have low parasite loads, since the prevalence and intensity of infection have declined after many years of successful control. Therefore, the low sensitivity of parasitological methods due to the low parasite loads, and the undefined specificity of immunological methods mainly because of cross-reactions with other parasitic infections, do not allow a proper diagnosis of the disease. Furthermore, in the case of past infection, antibodies may remain, yielding positive serological results with certain techniques, even though the patient has been treated (Alarco´n de Noya et al., 2007). Although the PCR technique has been utilized extensively for the diagnosis of numerous human diseases, its application for neglected diseases, especially schistosomiasis, has only recently been explored. Pontes et al., (2002) reported the first use of PCR for the diagnosis of S. mansoni DNA in human serum and faecal samples, using a very sensitive marker, the 121-bp highly repeated sequence (Hamburger et al., 1991). This sequence has about 600,000 copies per cell, equivalent to 10% of the
Optimal condition 10 pg– 10 ag 2.5 mM 150 mM 0.4 mM 0.75 U 638C 35
total DNA of each cell of the parasite. For all PCRs, a prior standardization, adaptation and evaluation of the reaction conditions for use of the technique in each laboratory is required. For the standardization of PCR for amplification of the 121-bp highly repeated sequence of S. mansoni amounts of template DNA were evaluated, and the optimal amount of DNA ranged between 100 picograms (pg) (10212 g) and 1 femtogram (fg) (10215 g). Consequently, 1 pg was taken as the amount of DNA to use in subsequent determinations (table 1). The optimum reagent concentrations with good amplifications can be seen in table 1. The optimal concentrations of dNTPs, primers, MgCl2 and Taq polymerase were lower than those used by Pontes et al. (2002), thereby allowing for a reduction in the use of reagents. Regarding the amplification program, where the annealing temperature and the number of cycles were evaluated, it was determined that at 638C more defined and non-contaminated bands were observed, while at 608C more tenuous bands and the presence of contamination in the negative controls were observed. In addition, using 30 cycles of amplification, no bands were observed, whereas when using 35 cycles, the diagnostic bands were evident (table 1). Schistosoma mansoni DNA was able to produce adequate amplification (sharp bands, with the absence of nonspecific products and smears) in a range from 10 pg to 10 ag (10218 g), with the analytical sensitivity of the technique at 10 ag, indicating the minimum amount of DNA amplified by PCR (table 1). The sensitivity of conventional PCR based on other targets, such as Schistosoma 28S rDNA or internal transcribed spacer (ITS) rDNA, ranged from 0.98 to 15 pg, showing PCR to be useful for the detection of S. mansoni infection and that it may be used in non-invasive samples such as urine and faeces. Furthermore, this technique is more sensitive than the two classical methods for the diagnosis of schistosomiasis, the Kato – Katz and enzyme-linked immunosorbent assay (ELISA) (Sandoval et al., 2006). According to Hamburger et al. (1998b), the minimum amount of S. mansoni DNA detected by the 121-bp highly repeated sequence of S. mansoni PCR technique was 1.2 fg. Similarly, Gomes et al. (2009) showed that the minimum amount of DNA detected by PCR was 3 fg. Comparing these results, with a sensitivity around 1 –3 fg, with our first PCR using the reaction conditions of Pontes et al. (2002), we obtained a result of 1 fg; but later, using the
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standardized PCR, the sensitivity increased to 10 ag. The S. mansoni genome contains around 580 fg of DNA, so 10 ag is equivalent to less than one-thousandth of the amount of DNA contained in a parasite egg or cell. This result suggests a very high sensitivity, the best obtained for molecular diagnosis of parasitic diseases using conventional PCR. This is an important factor for the diagnosis of the disease, since currently available methods show sensitivity problems because infected patients have low parasite loads. On the other hand, the high sensitivity of the standardized PCR could make it an important tool for the evaluation of treatment and as a criterion of cure. In Brazil, Pontes et al. (2002) showed that the high sensitivity of the method allowed detection of parasite DNA in stool samples containing only 2.4 eggs/g of faeces, making the PCR method ten times more sensitive than the Kato –Katz method. In the present study the standardized PCR method could, in theory, detect the amount of DNA corresponding to less than 1 egg/g of faeces. Compared with the diagnostic sensitivity of the Kato – Katz technique: according to Gomes et al. (2009), in their study population of 67 inhabitants of an endemic area in Brazil, the number of positive samples detected using the PCR technique was higher (61.2% prevalence) than that determined by the Kato – Katz technique (41.8%) in positive patients with low parasite loads. In addition, the amplification reaction showed that there was amplification only for DNA samples of S. mansoni, with no cross-reaction with the DNA of other helminths or protozoa, indicating a specificity of 100%. Similar results were obtained by Pontes et al. (2002, 2003), obtaining no amplification with DNA from the parasitic helminths Ascaris lumbricoides, Ancylostoma duodenale, T. solium and Trichuris trichiuria. Of 86 DNA samples extracted from sera of patients with immunological diagnosis of schistosomiasis, using the standardized PCR, only two were positive (fig. 1). All patients were negative using coprological examinations (Kato– Katz) and positive for Western blot, but only some patients showed positivity with ELISA and the circumoral precipitin test. All positive tests involved detection using circulating antibodies.
Therefore, PCR could assess whether there is current transmission in the geographical area or if circulating antibodies correspond to past infections due to immunological memory of each individual. The standardized PCR technique allowed the demonstration of active infections in two patients from this endemic area. The results showed a characteristic focus of low transmission in Venezuela, and that treatment during the previous year may have decreased transmission. In addition, serum may not be the most sensitive sample, because there is more likelihood of finding DNA in the stools. It would be important to apply the standardized PCR to stool samples, since in a recent study (Carneiro et al., 2013) the PCR results using stool samples from patients with positive and negative coprological and immunological diagnosis showed low diagnostic sensitivity. However, other studies have demostrated high sensitivity using this molecular target. The assessment of a single faecal sample by PCR detected more cases of infection than the analysis of two slides of one sample using the Kato – Katz technique, suggesting that PCR can be a useful diagnostic tool, particularly in areas with low endemicity (Costa et al., 2012). On the other hand, Wichmann et al. (2013), using sera, showed 92% diagnostic sensitivity of real-time PCR based on the 121-bp highly repeated sequence of S. mansoni. In addition, Enk et al. (2012), using urine, and Ha¨rter et al. (2014), using cerebrospinal fluid, obtained high sensitivity for both types of samples, suggesting that these were also useful in the diagnosis of schistosomiasis and neuroschistosomiasis, respectively. Additionally, for the early diagnosis of acute schistosomiasis, real-time PCR in serum is more sensitive than classic diagnostic tools such as serology or microscopy, irrespective of the region of infection (Wichmann et al., 2013). The present results show that the PCR technique for amplifying DNA from S. mansoni, besides being specific, is capable of detecting small amounts of parasite DNA, allowing early diagnosis of the disease. Therefore, this conventional PCR assay is a valuable alternative for the diagnosis of S. mansoni infections. Prevalence values using molecular diagnosis would validate serological diagnosis, and will probably be the gold standard technique for the diagnosis of schistosomiasis in situations of low transmission. Molecular diagnosis techniques for S. mansoni have never been used in Venezuela. The sensitive and accurate identification of human infection and the precise monitoring of schistosome transmission sites may be important tools for the long-term control of this disease.
References
Fig. 1. Detection of S. mansoni DNA by PCR in serum samples of patients. Lanes: 1, molecular marker 100 bp plus DNA ladder (Promega, Madison, Wisconsin, USA); 2, positive control; 3, negative control; 4–5, samples of positive patients; 6–7, samples of negative patients.
Abath, F., Gomes, A., Melo, F., Barbosa, C. & Werkhauser, R. (2006) Molecular approaches for the detection of Schistosoma mansoni: possible applications in the detection of snail infection, monitoring of transmission sites, and diagnosis of human infection. Memo´rias do Instituto Oswaldo Cruz 101 (suppl. 1), 145– 148. Alarco´n de Noya, B., Ruiz, R., Losada, S., Colmenares, C., Contreras, R., Cesari, I.M. & Noya, O. (2007) Detection
Molecular diagnosis of schistosomiasis
of schistosomiasis cases in low-transmission areas based on coprologic and serologic criteria: the Venezuelan experience. Acta Tropica 103, 41 – 49. Barreto, M. (1991) Geographical and socioeconomic factors relating to the distribution of Schistosoma mansoni infection in an urban area of north-east Brazil. Bulletin of the World Health Organization 69, 93 – 102. Carneiro, T.R., Peralta, R.H., Pinheiro, M.C., de Oliveira, S.M., Peralta, J.M. & Bezerra, F.S. (2013) A conventional polymerase chain reaction-based method for the diagnosis of human schistosomiasis in stool samples from individuals in a low-endemicity area. Memo´rias do Instituto Oswaldo Cruz 108, 1037– 1044. Cesari, I.M., Ballen, D.E., Mendoza, L. & Matos, C. (2005) Detection of Schistosoma mansoni membrane antigens by immunoblot analysis of sera of patients from low-transmission areas. Clinical and Diagnostic Laboratory Immunology 12, 280– 286. Costa, G.C., Dos Santos, L.H., Gomes, L.I., Rabello, A., Ribeiro, L.C., Gorza, K.K., Cerrato, S.H., Soares, E.S. & Abramo, C. (2012) Polymerase chain reaction for the evaluation of Schistosoma mansoni infection in two low endemicity areas of Minas Gerais, Brazil. Memo´rias do Instituto Oswaldo Cruz 107, 899– 902. Enk, M.J., Oliveira, G. & Rodrigues, N.B. (2012) Diagnostic accuracy and applicability of a PCR system for the detection of Schistosoma mansoni DNA in human urine samples from an endemic area. Public Library of Science 7, e38947. doi:10.1371/journal. pone.0038947. Gomes, L.I., Marques, L.H., Enk, M.J., Coelho, P.M. & Rabello, A. (2009) Further evaluation of an updated PCR assay for the detection of Schistosoma mansoni DNA in human stool samples. Memo´rias do Instituto Oswaldo Cruz 104, 1194– 1196. Hamburger, J., Turetzky, T., Kapeller, I. & Deresiewicz, R. (1991) Highly repeated short DNA sequences in the genome of Schistosoma mansoni recognized by a species specific probe. Molecular and Biochemical Parasitology 44, 73 – 80. Hamburger, J., He-Na, H., Xin, Y.X., Ramzy, R.M., Jourdane, J. & Ruppel, A. (1998a) A polymerase chain reaction assay for detecting snails infected with bilharzia parasites (Schistosoma mansoni) from very early prepatency. American Journal of Tropical Medicine and Hygiene 59, 872– 876.
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Hamburger, J., Xin, Y.X., Ramzy, R.M., Jourdane, J. & Ruppel, A. (1998b) Development and laboratory evaluation of a polymerase chain reaction for monitoring Schistosoma mansoni infestation of water. American Journal of Tropical Medicine and Hygiene 59, 468– 473. Ha¨rter, G., Frickmann, H., Zenk, S., Wichmann, D., Ammann, B., Kern, P., Fleischer, B., Tannich, E. & Poppert, S. (2014) Diagnosis of neuroschistosomiasis by antibody specificity index and semi-quantitative real-time PCR from cerebrospinal fluid and serum. Journal of Medical Microbiology 63, 309– 312. Pontes, L., Dias Neto, E. & Rabello, A. (2002) Detection by polymerase chain reaction of Schistosoma mansoni DNA in human serum and feces. American Journal of Tropical Medicine and Hygiene 66, 157– 162. Pontes, L., Oliveira, M., Katz, N., Dias Neto, M. & Rabello, A. (2003) Comparison of a polymerase chain reaction and the Kato – Katz technique for diagnosing infection with Schistosoma mansoni. American Journal of Tropical Medicine and Hygiene 68, 652– 656. Pujol, F.H. & Cesari, I.M. (1990) Antigenicity of adult Schistosoma mansoni alkaline phosphatase. Parasite Immunology 12, 189– 198. Pujol, F.H., Alarcon de Noya, B. & Cesari, I.M. (1989) Immunodiagnosis of schistosomiasis mansoni with APIA (alkaline phosphatase immunoassay). Immunological Investigation 18, 1071– 1080. Sambrook, J. & Russell, D. (2001) Molecular cloning. A laboratory manual. 3rd edn. New York, Cold Spring Harbor Laboratory Press. Sandoval, N., Siles-Lucas, M., Lopez, J., Pe´rez, J., Ga´rate, T. & Muro, A. (2006) Schistosoma mansoni: A diagnostic approach to detect acute schistosomiasis infection in a murine model by PCR. Experimental Parasitology 114, 84 – 88. WHO (2014) Schistosomiasis fact sheet no. 115. Available at website http://www.who.int/schistosomiasis/en/ (accessed 14 April 2014). Wichmann, D., Poppert, S., Von Thien, H., Clerinx, J., Dieckmann, S., Jensenius, M., Parola, P., Richter, J., Schunk, M., Stich, A., Zanger, P., Burchard, G.D. & Tannich, E. (2013) Prospective European-wide multicentre study on a blood based real-time PCR for the diagnosis of acute schistosomiasis. BioMed Central Infectious Diseases 13, 55. doi:10.1186/14712334-13-55.
doi:10.1017/S0022149X14000595
Polymerase chain reaction for the amplification of the 121-bp repetitive sequence of Schistosoma mansoni: a highly sensitive potential diagnostic tool for areas of low endemicity E. Ferrer1,2*, F. Pe´rez1, I. Bello1, A. Bolı´var1, M. Lares1, A. Osorio3, L. Leo´n3, M. Amarista4 and R.N. Incani5 1
Instituto de Investigaciones Biome´dicas ‘Dr. Francisco J. Triana Alonso’ (BIOMED), Facultad de Ciencias de la Salud, Universidad de Carabobo Sede Aragua, Maracay, Venezuela: 2Departamento de Parasitologı´a, Facultad de Ciencias de la Salud, Universidad de Carabobo Sede Aragua, Maracay, Venezuela: 3Programa Nacional de Prevencio´n y Control de Parasitosis Intestinales y Esquistosomosis, Direccio´n General de Salud Ambiental, Ministerio del Poder Popular Para La Salud, Maracay, Venezuela: 4Centro de Estudios de Enfermedades Ende´micas y Salud Ambiental (CEEESA), Instituto de Altos Estudios ‘Dr. Arnoldo Gabaldo´n’, Ministerio del Poder Popular Para La Salud, Maracay, Venezuela: 5Laboratorio de Investigaciones en Bilharzia, Departamento de Parasitologı´a, Facultad de Ciencias de la Salud, Sede Carabobo, Universidad de Carabobo, Valencia, Venezuela (Received 23 April 2014; Accepted 11 July 2014; First Published Online 20 August 2014) Abstract Schistosomiasis is a disease caused by parasitic flatworms of the genus Schistosoma, whose diagnosis has limitations, such as the low sensitivity and specificity of parasitological and immunological methods, respectively. In the present study an alternative molecular technique requiring previous standardization was carried out using the polymerase chain reaction (PCR) for the amplification of a 121-bp highly repetitive sequence for Schistosoma mansoni. DNA was extracted from eggs of S. mansoni by salting out. Different conditions were standardized for the PCR technique, including the concentration of reagents and the DNA template, annealing temperature and number of cycles, followed by the determination of the analytical sensitivity and specificity of the technique. Furthermore, the standardized PCR technique was employed in DNA extracted, using Chelexw100, from samples of sera of patients with an immunodiagnosis of schistosomiasis. The optimal conditions for the PCR were 2.5 mM MgCl2, 150 mM deoxynucleoside triphosphates (dNTPs), 0.4 mM primers, 0.75 U DNA polymerase, using 35 cycles and an annealing temperature of 638C.
*Fax: þ58 2435333 E-mail: [email protected]
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E. Ferrer et al.
The analytical sensitivity of the PCR was 10 attograms of DNA and the specificity was 100%. The DNA sequence was successfully detected in the sera of two patients, demonstrating schistosomiasis transmission, although low, in the community studied. The standardized PCR technique, using smaller amounts of reagents than in the original protocol, is highly sensitive and specific for the detection of DNA from S. mansoni and could be an important tool for diagnosis in areas of low endemicity.
Introduction
Materials and methods
Schistosomiasis is a disease caused by parasitic flatworms of the genus Schistosoma. The disease is a major public health problem, affecting more than 240 million people in developing countries. Schistosoma mansoni is endemic in 54 countries in South America, the Caribbean, Africa and the eastern Mediterranean region (WHO, 2014). Currently schistosomiasis is endemic in the Americas. It is a predominantly rural parasitosis, although it may be suburban and even urban, depending on the existence of the conditions for transmission. Socio-economic factors influence the transmission of schistosomiasis, being prevalent in tropical and subtropical areas, in poor communities without potable water and adequate sanitation (Barreto, 1991; WHO, 2014). The actual epidemiological situation of schistosomiasis in Venezuela is of low transmission (Alarco´n de Noya et al., 2007), which makes the parasitological diagnosis by standard coprology difficult. Serological diagnosis is carried out in Venezuela using the cumbersome goldstandard circumoval precipitin test (COPT), in addition to two other tests, the alkaline phosphatase immunoassay (Pujol et al., 1989; Pujol & Cesari, 1990) and the Western blot (Cesari et al., 2005) – both tests of experimental use, requiring a reliable validation test, since the prevalence value for both tests is much higher than that obtained by COPT. Molecular diagnosis using polymerase chain reaction (PCR) appears to be an alternative. The PCR systems described are sensitive and have the potential to detect parasite DNA in snails, monitor cercariae in water bodies (Hamburger et al., 1991, 1998a, b; Abath et al., 2006) and diagnose human infection (Pontes et al., 2002, 2003; Abath et al., 2006). More extensive validation studies in endemic areas are crucial to investigate the suitability of this molecular tool for the diagnosis of infected humans. Standardization of a protocol for PCR amplification of the 121-bp highly repetitive sequence of S. mansoni (Pontes et al., 2002) could provide a technique that allows rapid, accurate, sensitive and specific diagnosis, which could be used for both clinical diagnosis and epidemiological studies, being able to distinguish active from non-active foci, a fact not easily attainable using immunological methods since antibodies may persist over time, while the presence of parasite DNA depends on an active infection. In addition, making the proper diagnosis of active outbreaks can provide benefits for the treatment of infected humans, and surveillance of infection in vectors and waters will be more reliable for decisions to be taken by a Schistosomiasis Control Programme.
Collection of samples In order to standardize protocols of the PCR for the amplification of a 121-bp highly repeated sequence of S. mansoni (Pontes et al., 2002), egg samples of the Brazilian BH strain of S. mansoni obtained from experimentally infected golden hamsters were used. In addition, DNA samples of other parasites, such as Fasciola hepatica, Taenia solium, Taenia saginata, Toxocara canis, Trypanosoma cruzi, Leishmania chagasi and Toxoplasma gondii, and human DNA were used to determine the specificity of PCR. In addition, serum samples from 86 humans with immunological diagnosis of schistosomiasis from an ancient endemic focus of this disease (Barrio Nuevo, Tiara, Aragua State) were analysed. The patients were treated 1 year previously with praziquantel, and evaluated a year after treatment. The results showed that all patients were negative by coprological diagnosis (Kato – Katz) and positive by immunological diagnosis (Western blot). These serum samples were used to evaluate if there was active transmission at the time of evaluation, or if the immunological results were due to past infections. Molecular analysis Conventional methods for the extraction of DNA, such as extraction by salting out, and the use of Chelexw 100 resin (BioRad Laboratories, Redmond, Washington, USA) were used. DNA extraction by the salting-out technique was performed according to the original protocol described by Sambrook & Russell (2001), but increasing the time of incubation with proteinase K to 4 h and the centrifugation time after incubating with proteinase K to 30 min, to match the type of sample (parasite eggs). DNA extraction from serum samples from 86 patients with immunological diagnosis of schistosomiasis, using resin Chelexw 100, was done following the instructions of the manufacturer. The PCR for the amplification of the 121-bp highly repeated sequence of S. mansoni (Pontes et al., 2002) was carried out using DNA samples extracted from parasite eggs obtained from the liver of experimentally infected golden hamsters. The amplification reactions were carried out according to the protocol described by Pontes et al., 2002), using the forward 50 -GATCTGAATCCGACCAACCG-30 and reverse 50 -ATATTAACGCCCACGCTCTC-30 primers and varying the concentrations of
771
Molecular diagnosis of schistosomiasis Table 1. The concentration and quantity of reagents, including conditions tested, for the PCR amplification of the 121-bp repetitive sequence of S. mansoni. Reagent/condition
Quantity/concentration/condition tested
DNA
1 mg, 100 ng, 10 ng, 1 ng, 100 pg, 10 pg, 1 pg, 100 fg, 10 fg, 1 fg, 100 ag, 10 ag, 1 ag, 0.1 ag 1 mM , 1.5 mM , 2 mM , 2.5 mM 100 mM , 150 mM , 200 mM , 250 mM 0.2, 0.4, 0.6, 0.8 mM 0.5 U, 0.75 U, 1.0 U, 1.25 U 608C, 638C 30, 35
Magnesium chloride dNTPs Primers Taq DNA polymerase Annealing temperature Number of cycles
reagents, such as deoxynucleoside triphosphates (dNTPs), primers, polymerase and magnesium chloride, and conditions of temperatures and number of cycles (table 1), to verify the conditions for the best amplification. PCR products were visualized in 2% agarose gels and compared with molecular size markers. All tested conditions for PCR were compared, and they were selected as optimal, where the expected bands were clear and with the absence of non-specific products or smears, observed through photographic images of agarose gels. The analytical sensitivity was determined using different quantities of DNA, 1 mg to 0.1 attogram (at) (table 1). The specificity was also determined using DNA samples from other parasites, such as F. hepatica, T. solium, T. saginata, T. canis, T.cruzi, L. chagasi and T. gondii, and human DNA. The previously standardized PCR for the amplification of the 121-bp highly repeated sequence of S. mansoni (Pontes et al., 2002) was performed using DNA samples extracted from serum samples from 86 patients with immunological diagnosis of schistosomiasis.
Results and discussion In Venezuela, transmission of S. mansoni can be characterized as hypoendemic and infected patients have low parasite loads, since the prevalence and intensity of infection have declined after many years of successful control. Therefore, the low sensitivity of parasitological methods due to the low parasite loads, and the undefined specificity of immunological methods mainly because of cross-reactions with other parasitic infections, do not allow a proper diagnosis of the disease. Furthermore, in the case of past infection, antibodies may remain, yielding positive serological results with certain techniques, even though the patient has been treated (Alarco´n de Noya et al., 2007). Although the PCR technique has been utilized extensively for the diagnosis of numerous human diseases, its application for neglected diseases, especially schistosomiasis, has only recently been explored. Pontes et al., (2002) reported the first use of PCR for the diagnosis of S. mansoni DNA in human serum and faecal samples, using a very sensitive marker, the 121-bp highly repeated sequence (Hamburger et al., 1991). This sequence has about 600,000 copies per cell, equivalent to 10% of the
Optimal condition 10 pg– 10 ag 2.5 mM 150 mM 0.4 mM 0.75 U 638C 35
total DNA of each cell of the parasite. For all PCRs, a prior standardization, adaptation and evaluation of the reaction conditions for use of the technique in each laboratory is required. For the standardization of PCR for amplification of the 121-bp highly repeated sequence of S. mansoni amounts of template DNA were evaluated, and the optimal amount of DNA ranged between 100 picograms (pg) (10212 g) and 1 femtogram (fg) (10215 g). Consequently, 1 pg was taken as the amount of DNA to use in subsequent determinations (table 1). The optimum reagent concentrations with good amplifications can be seen in table 1. The optimal concentrations of dNTPs, primers, MgCl2 and Taq polymerase were lower than those used by Pontes et al. (2002), thereby allowing for a reduction in the use of reagents. Regarding the amplification program, where the annealing temperature and the number of cycles were evaluated, it was determined that at 638C more defined and non-contaminated bands were observed, while at 608C more tenuous bands and the presence of contamination in the negative controls were observed. In addition, using 30 cycles of amplification, no bands were observed, whereas when using 35 cycles, the diagnostic bands were evident (table 1). Schistosoma mansoni DNA was able to produce adequate amplification (sharp bands, with the absence of nonspecific products and smears) in a range from 10 pg to 10 ag (10218 g), with the analytical sensitivity of the technique at 10 ag, indicating the minimum amount of DNA amplified by PCR (table 1). The sensitivity of conventional PCR based on other targets, such as Schistosoma 28S rDNA or internal transcribed spacer (ITS) rDNA, ranged from 0.98 to 15 pg, showing PCR to be useful for the detection of S. mansoni infection and that it may be used in non-invasive samples such as urine and faeces. Furthermore, this technique is more sensitive than the two classical methods for the diagnosis of schistosomiasis, the Kato – Katz and enzyme-linked immunosorbent assay (ELISA) (Sandoval et al., 2006). According to Hamburger et al. (1998b), the minimum amount of S. mansoni DNA detected by the 121-bp highly repeated sequence of S. mansoni PCR technique was 1.2 fg. Similarly, Gomes et al. (2009) showed that the minimum amount of DNA detected by PCR was 3 fg. Comparing these results, with a sensitivity around 1 –3 fg, with our first PCR using the reaction conditions of Pontes et al. (2002), we obtained a result of 1 fg; but later, using the
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standardized PCR, the sensitivity increased to 10 ag. The S. mansoni genome contains around 580 fg of DNA, so 10 ag is equivalent to less than one-thousandth of the amount of DNA contained in a parasite egg or cell. This result suggests a very high sensitivity, the best obtained for molecular diagnosis of parasitic diseases using conventional PCR. This is an important factor for the diagnosis of the disease, since currently available methods show sensitivity problems because infected patients have low parasite loads. On the other hand, the high sensitivity of the standardized PCR could make it an important tool for the evaluation of treatment and as a criterion of cure. In Brazil, Pontes et al. (2002) showed that the high sensitivity of the method allowed detection of parasite DNA in stool samples containing only 2.4 eggs/g of faeces, making the PCR method ten times more sensitive than the Kato –Katz method. In the present study the standardized PCR method could, in theory, detect the amount of DNA corresponding to less than 1 egg/g of faeces. Compared with the diagnostic sensitivity of the Kato – Katz technique: according to Gomes et al. (2009), in their study population of 67 inhabitants of an endemic area in Brazil, the number of positive samples detected using the PCR technique was higher (61.2% prevalence) than that determined by the Kato – Katz technique (41.8%) in positive patients with low parasite loads. In addition, the amplification reaction showed that there was amplification only for DNA samples of S. mansoni, with no cross-reaction with the DNA of other helminths or protozoa, indicating a specificity of 100%. Similar results were obtained by Pontes et al. (2002, 2003), obtaining no amplification with DNA from the parasitic helminths Ascaris lumbricoides, Ancylostoma duodenale, T. solium and Trichuris trichiuria. Of 86 DNA samples extracted from sera of patients with immunological diagnosis of schistosomiasis, using the standardized PCR, only two were positive (fig. 1). All patients were negative using coprological examinations (Kato– Katz) and positive for Western blot, but only some patients showed positivity with ELISA and the circumoral precipitin test. All positive tests involved detection using circulating antibodies.
Therefore, PCR could assess whether there is current transmission in the geographical area or if circulating antibodies correspond to past infections due to immunological memory of each individual. The standardized PCR technique allowed the demonstration of active infections in two patients from this endemic area. The results showed a characteristic focus of low transmission in Venezuela, and that treatment during the previous year may have decreased transmission. In addition, serum may not be the most sensitive sample, because there is more likelihood of finding DNA in the stools. It would be important to apply the standardized PCR to stool samples, since in a recent study (Carneiro et al., 2013) the PCR results using stool samples from patients with positive and negative coprological and immunological diagnosis showed low diagnostic sensitivity. However, other studies have demostrated high sensitivity using this molecular target. The assessment of a single faecal sample by PCR detected more cases of infection than the analysis of two slides of one sample using the Kato – Katz technique, suggesting that PCR can be a useful diagnostic tool, particularly in areas with low endemicity (Costa et al., 2012). On the other hand, Wichmann et al. (2013), using sera, showed 92% diagnostic sensitivity of real-time PCR based on the 121-bp highly repeated sequence of S. mansoni. In addition, Enk et al. (2012), using urine, and Ha¨rter et al. (2014), using cerebrospinal fluid, obtained high sensitivity for both types of samples, suggesting that these were also useful in the diagnosis of schistosomiasis and neuroschistosomiasis, respectively. Additionally, for the early diagnosis of acute schistosomiasis, real-time PCR in serum is more sensitive than classic diagnostic tools such as serology or microscopy, irrespective of the region of infection (Wichmann et al., 2013). The present results show that the PCR technique for amplifying DNA from S. mansoni, besides being specific, is capable of detecting small amounts of parasite DNA, allowing early diagnosis of the disease. Therefore, this conventional PCR assay is a valuable alternative for the diagnosis of S. mansoni infections. Prevalence values using molecular diagnosis would validate serological diagnosis, and will probably be the gold standard technique for the diagnosis of schistosomiasis in situations of low transmission. Molecular diagnosis techniques for S. mansoni have never been used in Venezuela. The sensitive and accurate identification of human infection and the precise monitoring of schistosome transmission sites may be important tools for the long-term control of this disease.
References
Fig. 1. Detection of S. mansoni DNA by PCR in serum samples of patients. Lanes: 1, molecular marker 100 bp plus DNA ladder (Promega, Madison, Wisconsin, USA); 2, positive control; 3, negative control; 4–5, samples of positive patients; 6–7, samples of negative patients.
Abath, F., Gomes, A., Melo, F., Barbosa, C. & Werkhauser, R. (2006) Molecular approaches for the detection of Schistosoma mansoni: possible applications in the detection of snail infection, monitoring of transmission sites, and diagnosis of human infection. Memo´rias do Instituto Oswaldo Cruz 101 (suppl. 1), 145– 148. Alarco´n de Noya, B., Ruiz, R., Losada, S., Colmenares, C., Contreras, R., Cesari, I.M. & Noya, O. (2007) Detection
Molecular diagnosis of schistosomiasis
of schistosomiasis cases in low-transmission areas based on coprologic and serologic criteria: the Venezuelan experience. Acta Tropica 103, 41 – 49. Barreto, M. (1991) Geographical and socioeconomic factors relating to the distribution of Schistosoma mansoni infection in an urban area of north-east Brazil. Bulletin of the World Health Organization 69, 93 – 102. Carneiro, T.R., Peralta, R.H., Pinheiro, M.C., de Oliveira, S.M., Peralta, J.M. & Bezerra, F.S. (2013) A conventional polymerase chain reaction-based method for the diagnosis of human schistosomiasis in stool samples from individuals in a low-endemicity area. Memo´rias do Instituto Oswaldo Cruz 108, 1037– 1044. Cesari, I.M., Ballen, D.E., Mendoza, L. & Matos, C. (2005) Detection of Schistosoma mansoni membrane antigens by immunoblot analysis of sera of patients from low-transmission areas. Clinical and Diagnostic Laboratory Immunology 12, 280– 286. Costa, G.C., Dos Santos, L.H., Gomes, L.I., Rabello, A., Ribeiro, L.C., Gorza, K.K., Cerrato, S.H., Soares, E.S. & Abramo, C. (2012) Polymerase chain reaction for the evaluation of Schistosoma mansoni infection in two low endemicity areas of Minas Gerais, Brazil. Memo´rias do Instituto Oswaldo Cruz 107, 899– 902. Enk, M.J., Oliveira, G. & Rodrigues, N.B. (2012) Diagnostic accuracy and applicability of a PCR system for the detection of Schistosoma mansoni DNA in human urine samples from an endemic area. Public Library of Science 7, e38947. doi:10.1371/journal. pone.0038947. Gomes, L.I., Marques, L.H., Enk, M.J., Coelho, P.M. & Rabello, A. (2009) Further evaluation of an updated PCR assay for the detection of Schistosoma mansoni DNA in human stool samples. Memo´rias do Instituto Oswaldo Cruz 104, 1194– 1196. Hamburger, J., Turetzky, T., Kapeller, I. & Deresiewicz, R. (1991) Highly repeated short DNA sequences in the genome of Schistosoma mansoni recognized by a species specific probe. Molecular and Biochemical Parasitology 44, 73 – 80. Hamburger, J., He-Na, H., Xin, Y.X., Ramzy, R.M., Jourdane, J. & Ruppel, A. (1998a) A polymerase chain reaction assay for detecting snails infected with bilharzia parasites (Schistosoma mansoni) from very early prepatency. American Journal of Tropical Medicine and Hygiene 59, 872– 876.
773
Hamburger, J., Xin, Y.X., Ramzy, R.M., Jourdane, J. & Ruppel, A. (1998b) Development and laboratory evaluation of a polymerase chain reaction for monitoring Schistosoma mansoni infestation of water. American Journal of Tropical Medicine and Hygiene 59, 468– 473. Ha¨rter, G., Frickmann, H., Zenk, S., Wichmann, D., Ammann, B., Kern, P., Fleischer, B., Tannich, E. & Poppert, S. (2014) Diagnosis of neuroschistosomiasis by antibody specificity index and semi-quantitative real-time PCR from cerebrospinal fluid and serum. Journal of Medical Microbiology 63, 309– 312. Pontes, L., Dias Neto, E. & Rabello, A. (2002) Detection by polymerase chain reaction of Schistosoma mansoni DNA in human serum and feces. American Journal of Tropical Medicine and Hygiene 66, 157– 162. Pontes, L., Oliveira, M., Katz, N., Dias Neto, M. & Rabello, A. (2003) Comparison of a polymerase chain reaction and the Kato – Katz technique for diagnosing infection with Schistosoma mansoni. American Journal of Tropical Medicine and Hygiene 68, 652– 656. Pujol, F.H. & Cesari, I.M. (1990) Antigenicity of adult Schistosoma mansoni alkaline phosphatase. Parasite Immunology 12, 189– 198. Pujol, F.H., Alarcon de Noya, B. & Cesari, I.M. (1989) Immunodiagnosis of schistosomiasis mansoni with APIA (alkaline phosphatase immunoassay). Immunological Investigation 18, 1071– 1080. Sambrook, J. & Russell, D. (2001) Molecular cloning. A laboratory manual. 3rd edn. New York, Cold Spring Harbor Laboratory Press. Sandoval, N., Siles-Lucas, M., Lopez, J., Pe´rez, J., Ga´rate, T. & Muro, A. (2006) Schistosoma mansoni: A diagnostic approach to detect acute schistosomiasis infection in a murine model by PCR. Experimental Parasitology 114, 84 – 88. WHO (2014) Schistosomiasis fact sheet no. 115. Available at website http://www.who.int/schistosomiasis/en/ (accessed 14 April 2014). Wichmann, D., Poppert, S., Von Thien, H., Clerinx, J., Dieckmann, S., Jensenius, M., Parola, P., Richter, J., Schunk, M., Stich, A., Zanger, P., Burchard, G.D. & Tannich, E. (2013) Prospective European-wide multicentre study on a blood based real-time PCR for the diagnosis of acute schistosomiasis. BioMed Central Infectious Diseases 13, 55. doi:10.1186/14712334-13-55.
