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Diagnosis of infections with Leishmania infantum using PCR–ELISA

  • J. MARTIN-SANCHEZ (a1), M. C. LOPEZ-LOPEZ (a2), C. ACEDO-SANCHEZ (a1), J. J. CASTRO-FAJARDO (a3), J. A. PINEDA (a4) and F. MORILLAS-MARQUEZ (a1)...

Abstract

On the basis of partial amplification of a cloned fragment of kDNA of Leishmania infantum which is specific for this species, we developed a PCR–ELISA technique which avoids the problems associated with classical diagnostic techniques. This technique was tested on 33 L. infantum strains from 19 different zymodemes, which were recognized equally. It was also used on human and canine clinical samples. PCR–ELISA has a higher sensitivity than the other techniques used (IFAT, parasite cultures, optical microscopy of stained samples) and permits detection of a minimum of 0.1 promastigotes or 1 fg of genomic DNA. PCR–ELISA can be used to diagnose human cutaneous leishmaniasis using material obtained by scraping the lesion margin, and human visceral leishmaniasis in HIV(+) individuals and canine leishmaniasis with peripheral blood samples. The presence of L. infantum in dogs with low antibody titres with IFAT technique (20 and 40) was demonstrated indicating that seroprevalence data from epidemiological studies underestimate the true rates of infection.

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Corresponding author

Corresponding author: Departamento de Parasitología, Facultad de Farmacia, C/Manuel Clavero s/n, Campus Universitario de Cartuja, 18.071, Granada, Spain. Tel: 0034 958 243857. Fax: 0034 958 243862. E-mail: joaquina@platon.ugr.es

References

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ABRANCHES, P., LOPES, F. J., SILVA, F. M. C., RIBIERO, M. M. S. & PIRES, C. A. (1983). Le kala-azar au Portugal. III. Résultats d'une enquête sur la leishmaniose canine réalisée dans les environs de Lisbonne. Comparison des zones urbaines et rurales. Annales Parasitologie Humaine et Comparée 58, 307315.
ACEDO-SANCHEZ, C., MARTIN-SANCHEZ, J., VELEZ-BERNAL, I. D., SANCHIS-MARIN, M. C., LOUASSINI, M., MALDONADO, J. A. & MORILLAS-MARQUEZ, F. (1996). Leishmaniasis eco-epidemiology in the Alpujarra region (Granada province, southern Spain). International Journal for Parasitology 25, 303310.
ANDRESEN, K., GAAFAR, A., EL-HASSAN, A. M., ISMAIL, A., DAFALLA, M., THEANDER, T. G. & KHARAZMI, A. (1996). Evaluation of the polymerase chain reaction in the diagnosis of cutaneous leishmaniasis due to Leishmania major: a comparison with direct microscopy of smears and sections from lesions. Transactions of the Royal Society of Tropical Medicine and Hygiene 90, 133135.
BETTINI, S. & GRADONI, L. (1986). Canine leishmaniasis in the Mediterranean area and its implications for human leishmaniasis. Insect Science and its Applications 7, 241245.
DELGADO, O., GUEVARA, P., SILVA, S., BELFORT, E. & RAMIREZ, J. L. (1996). Follow-up of a human accidental infection by Leishmania (Viannia) braziliensis using conventional immunological techniques and polymerase chain-reaction. American Journal of Tropical Medicine and Hygiene 55, 267272.
D'OLIVEIRA, C., VAN DER WEIDE, M., HABELA, M. A., JACQUIET, P. & JONGEJAN, F. (1995). Detection of Theileria annulata in blood samples of carrier cattle by PCR. Journal of Clinical Microbiology 33, 26652669.
FISA, R., PORTUS, M., GALLEGO, M., VALLAS, D. & AISA, M. J. (1992). El diagnóstico serológico de la leishmaniosis canina en la comarca del Priorat (Tarragona). Clínica Veterinaria de pequeños animales 12, 3338.
GRAMICCIA, M. & GRADONI, L. (1989). Successful in vitro isolation and cultivation of italian dermotropic strains of Leishmania infantum sensu lato. Transactions of the Royal Society of Tropical Medicine and Hygiene 83, 76.
GRAMICCIA, M., GRADONI, L. & POZIO, E. (1987). Leishmania infantum sensu lato as an agent of cutaneous leishmaniasis in Abruzzi region (Italy). Transactions of the Royal Society of Tropical Medicine and Hygiene 81, 235237.
GRAMICCIA, M., SMITH, D. F., ANGELICI, M. C., READY, P. D. & GRADONI, L. (1992). A kinetoplast DNA probe diagnostic for Leishmania infantum. Parasitology 105, 2934.
JAMBOU, D., MARTY, P., JAMBOU, R., LE FICHOUX, Y., HAAS, P., JOURDAIN, N. & BAYADA, M. (1986). Preliminary serological study on canine leishmaniasis in the Alpes Maritimes Department, France. Transactions of the Royal Society of Tropical Medicine and Hygiene 80, 666667.
LANOTTE, G., RIOUX, J. A., CROSET, H. & VOLHARD, Y. (1978). Ecologie des Leishmanioses dans le Sud de la France. Les méthodes d'echantillonnage dans le dépistage et l'analyse de l'enzootie canine. Annales de Parasitologie Humaine et Comparée 53, 3345.
LASKAY, T., MIKO, T. L., NEGRESSE, Y., SOLBACH, W., ROLLINGHOFF, M. & FROMMEL, D. (1995). Detection of cutaneous Leishmania infection in paraffin-embedded skin biopsies using the polymerase chain reaction. Transactions of the Royal Society of Tropical Medicine and Hygiene 89, 273275.
LOPEZ-VELEZ, R., LAGUNA, F., ALVAR, J., PEREZ-MOLINA, J. A., MOLINA, R., MARTINEZ, P. & VILLARRUBIA, J. (1995). Parasitic culture of buffy coat for diagnosis of visceral leishmaniasis in human immodeficiency virus-infected patients. Journal of Clinical Microbiology 33, 937939.
MARTIN-SANCHEZ, J., GRAMICCIA, M., ANGELICI, M. C., ACEDO-SANCHEZ, C. & MORILLAS-MARQUEZ, F. (1998). Evaluation of the PLiD2-196bp kDNA probe specific for Leishmania infantum for the diagnosis of canine leishmaniasis in a focus of southern Spain. Research and Reviews in Parasitology 58, 9194.
MARTIN-SANCHEZ, J., LEPE, J. A., TOLEDO, A., UBEDA, J. M., GUEVARA, D. C., MORILLAS-MARQUEZ, F. & GRAMICCIA, M. (1999). Leishmania infantum enzymatic variants causing canine leishmaniasis in the Huelva province (south-west Spain). Transactions of the Royal Society of Tropical Medicine and Hygiene 93, 495496.
MARTIN-SANCHEZ, J., MORILLAS-MARQUEZ, F., SANCHIZ-MARIN, M. C. & ACEDO-SANCHEZ, C. (1994). Isoenzymatic characterization of the etiologic agent of canine leishmaniasis in the Granada region of southern Spain. American Journal of Tropical Medicine and Hygiene 50, 758762.
MARTIN-SANCHEZ, J., RUIZ-MARTINEZ, F., SALINAS-MARTINEZ DE LECEA, J. M., SANCHEZ-RABASCO, C., ACEDO-SANCHEZ, C., SANCHIZ-MARIN, M. C., DELGADO-FLORENCIO, V. & MORILLAS-MARQUEZ, F. (1996). Leishmania infantum Nicolle, 1908 from southern Spain: Characterisation of the strains from human visceral and cutaneous leishmaniasis and from sandflies; with a numerical analysis of the isoenzymatic data. Systematic Parasitology 33, 177182.
MARTINEZ-CRUZ, S., MARTINEZ-MORENO, A., MARTINEZ-MORENO, F. J., MARTINEZ-GÓMEZ, F. & HERNANDEZ-RODRIGUEZ, S. (1990). Epidemiología de la Leishmaniosis canina en Córdoba. Revista Ibérica de Parasitologia 50, 17.
MATHIS, A. & DEPLAZES, P. (1995). PCR and in vitro cultivation for detection of Leishmania spp. In diagnostic samples from humans and dogs. Journal of Clinical Microbiology 33, 11451149.
MORILLAS, F., SANCHEZ-RABASCO, F., OCAÑA, J., MARTIN-SANCHEZ, J., OCAÑA-WIHELMI, J., ACEDO, C. & SANCHIZ-MARIN, M. C. (1996). Leishmaniosis in the focus of the Axarquía region, Malaga province, southern Spain: a survey of the human, dog, and vector. Parasitology Research 82, 569570.
NIETO, C. G., NAVARRETE, M., HABELA, M. & HERNANDEZ-RODRIGUEZ, S. (1992). Seroprevalence of canine leishmaniasis around Cáceres, Spain. Preventive Veterinary Medicine 13, 173178.
NOYES, H. A., REYBURN, H., BAILEY, J. W. & SMITH, D. (1998). A nested-PCR-based schizodeme method for identifying Leishmania kinetoplast minicircle classes directly from clinical samples and its application to the study of the epidemiology of Leishmania tropica in Pakistan. Journal of Clinical Microbiology 36, 28772881.
PIARROUX, R., AZAIEZ, R., LOSSI, A. M., REYNIER, P., MUSCATELLI, F., GAMBARELLI, F., FONTES, M., DUMON, H. & QUILICI, M. (1993). Isolation and characterization of a repetitive DNA sequence from Leishmania infantum: development of a visceral leishmaniasis polymerase chain reaction. American Journal of Tropical Medicine and Hygiene 49, 364369.
PIARROUX, R., FONTES, M., PERASSO, R., GAMBARELLI, F., JOBLET, C., DUMON, H. & QUILICI, M. (1995). Phylogenetic relationships between Old World Leishmania strains revealed by analysis of a repetitive DNA sequence. Molecular and Biochemical Parasitology 32, 746749.
QUIAO, Z., MILES, M. A. & WILSON, S. M. (1995). Detection of parasites of the Leishmania donovani complex by a polymerase chain reaction-solution hybridization enzyme-linked immunoassay (PCR–SHELA). Parasitology 110, 269275.
RAMOS, A., MASLOV, D. A., FERNANDES, O., CAMPBELL, D. A. & SIMPSON, L. (1996). Detection and identification of human pathogenic Leishmania and Trypanosoma species by hybridization of PCR-amplified mini-exon repeats. Experimental Parasitology 82, 242250.
ULIANA, S. R. B., NELSON, K., BEVERLEY, S. M., CAMARGO, E. P. & LOETER-WINTER, L. M. (1994). Discrimination amongst Leishmania by polymerase chain reaction and hybridization with small subunit ribosomal DNA derived oligonucleotides. Journal of Eukaryotic Microbiology 41, 324330.
SAMBROOK, J., FRITSCH, E. F. & MANIATIS, T. (1989). Molecular Cloning. A Laboratory Manual, 2nd Edn. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, USA.
SEMIAO-SANTOS, S. J., EL HARITH, A., FERREIRA, E., PIRES, C. A., SOUSA, C. & GUSMAO, R. (1995). Evora district as a new focus for canine leishmaniasis in Portugal. Parasitology Research 81, 235239.
WORLD HEALTH ORGANIZATION (1989). Handbook on isolation, characterization and cryopreservation of Leishmania. UNDP/World Bank/WHO Special Programme for Research and Training in Tropical Diseases. Ed. David Evans. WHO, Geneva.
WORLD HEALTH ORGANIZATION (1996). Epidemiological analysis of 692 retrospective cases of Leishmania/HIV co-infection. World Health Organization. Division of Control of Tropical Diseases. WHO/LEISH/96.39. WHO, Geneva.

Keywords

Diagnosis of infections with Leishmania infantum using PCR–ELISA

  • J. MARTIN-SANCHEZ (a1), M. C. LOPEZ-LOPEZ (a2), C. ACEDO-SANCHEZ (a1), J. J. CASTRO-FAJARDO (a3), J. A. PINEDA (a4) and F. MORILLAS-MARQUEZ (a1)...

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