Polymorphism-specific PCR enhances the diagnostic performance of American tegumentary leishmaniasis and allows the rapid identification of Leishmania species from Argentina

BMC Infectious Diseases

Yoshihisa Hashiguchi¹  Miguel A Basombrío⁵  Masataka Korenaga⁶  Taketoshi Taniguchi³  Luis A Parada⁵  Julio R Nasser⁴  S Pamela Cajal⁴  María C Mora⁵  Ayako Tomatani³  Fabricio M Locatelli⁶  Tatsuyuki Mimori²  Paola A Barroso⁶  Jorge D Marco⁶ 

[1] Prometeo Proyecto, Centro de Biomedicina, Universidad Central del Ecuador, Quito, Ecuador;Department of Microbiology, School of Health Sciences, Kumamoto University, Kumamoto, Japan;Division of Molecular Biology, Science Research Center, Kochi University, Nankoku, Japan;Instituto de Investigaciones en Enfermedades Tropicales, Sede Regional Orán, Universidad Nacional de Salta, San Ramón de la Nueva Orán, Salta, Argentina;Instituto de Patología Experimental, Facultad de Ciencias de la Salud, Universidad Nacional de Salta / Consejo Nacional de Investigaciones Científicas y Técnicas, Salta, Argentina;Department of Parasitology, Kochi Medical School, Kochi University, Nankoku, Japan

关键词: Leishmania strains;    Species identification;    Argentina;    Diagnosis;    PS-PCR;    Leishmania panamensis;    Leishmania guyanensis;    Leishmania braziliensis;    Tegumentary leishmaniasis;    Leishmaniasis;   

Others  :  1159717
DOI  :  10.1186/1471-2334-12-191

 received in 2012-06-15, accepted in 2012-08-08,  发布年份 2012

【 摘 要 】

Background

The diagnosis of the leishmaniases poses enormous challenges in Argentina. The Polymorphism-Specific PCR (PS-PCR) designed and validated in our laboratories has been proven effective for typifying the Leishmania genus from cultured material. Here we evaluated the performance of this method in the diagnosis of American tegumentary leishmaniasis (ATL) and the rapid identification of Leishmania spp. directly from clinical specimens.

Methods

A total of 63 patients from northwestern Argentina, with cutaneous or mucocutaneous lesions, underwent an ATL diagnosis protocol which included clinical examination, Leishmanin skin test, and microscopic examination of dermal smears. In addition, we performed PS-PCR on DNA directly extracted from the specimens scraped from the lesions.

Results

Out of the 63 patients, 44 were classified as ATL cases and 19 as non-ATL cases. The diagnostic sensitivity of the microscopic analysis of dermal smears and PS-PCR individually were 70.5% and 81%, respectively. When performing both tests in parallel, this parameter increased significantly to 97.6% (p = 0.0018). The specificities, on the other hand, were 100%, 84.2%, and 83.3% for the combination, respectively (p > 0.05). Using the PS-PCR analysis we successfully identified the Leishmania spp. in 31 out of the 44 ATL cases. Twenty-eight (90.3%) cases were caused by L. (V.) braziliensis, two (6.5%) by L. (V.) guyanensis, and one (3.2%) by L. (V.) panamensis.

Conclusions

The efficacy of the ATL diagnosis was significantly improved by combining the dermal smear examination with a PS-PCR analysis. Our strategy allowed us to reach the diagnosis of ATL with high accuracy regarding the species of the etiological agent in 70.5% of the cases. Moreover, we diagnosed two cases of the disseminated cutaneous form caused by L. (V.) braziliensis and a cutaneous case due to L. (V.) panamensis infection, both findings reported for the first time in Argentina.

【 授权许可】

   
2012 Marco et al.; licensee BioMed Central Ltd.

【 图 表 】

Figure 1.

Figure 2.

【 参考文献 】

• [1]: Report of the Scientific Workving Group meeting on Leishmaniasis. World Health Organization, Geneva; 2004:5-16.
• [2]Salomon OD, Quintana MG, Zaidenberg M: Urban distribution of Phlebotominae in a cutaneous leishmaniasis focus, Argentina. Mem Inst Oswaldo Cruz 2008, 103(3):282-287.
• [3]Sosa Estani S, Campanini A, Sinagra A, Luna C, Peralta M, Coutada V, Medina L, Riarte A, Salomon D, Gomez A, et al.: Clinical features and diagnosis of mucocutaneous leishmaniasis in patients of an endemic area in Salta. Medicina (B Aires) 1998, 58(6):685-691.
• [4]Frank FM, Fernandez MM, Taranto NJ, Cajal SP, Margni RA, Castro E, Thomaz-Soccol V, Malchiodi EL: Characterization of human infection by Leishmania spp. in the Northwest of Argentina: immune response, double infection with Trypanosoma cruzi and species of Leishmania involved. Parasitology 2003, 126(Pt 1):31-39.
• [5]Marco JD, Barroso PA, Calvopina M, Kumazawa H, Furuya M, Korenaga M, Cajal SP, Mora MC, Rea MM, Borda CE, et al.: Species assignation of Leishmania from human and canine American tegumentary leishmaniasis cases by multilocus enzyme electrophoresis in North Argentina. Am J Trop Med Hyg 2005, 72(5):606-611.
• [6]Gil JF, Hoyos CL, Cimino RO, Krolewiecki AJ, Lopez Quiroga I, Cajal SP, Juarez M, Garcia Bustos MF, Mora MC, Marco JD, et al.: Role of three ELISA tests using promastigote homogenates of Leishmania braziliensis, L. amazonensis and L. guyanensis in the diagnosis of tegumentary leishmaniasis. Medicina (B Aires) 2011, 71(5):420-428.
• [7]Reithinger R, Dujardin JC: Molecular diagnosis of leishmaniasis: current status and future applications. J Clin Microbiol 2007, 45(1):21-25.
• [8]Mimori T, Sasaki J, Nakata M, Gomez EA, Uezato H, Nonaka S, Hashiguchi Y, Furuya M, Saya H: Rapid identification of Leishmania species from formalin-fixed biopsy samples by polymorphism-specific polymerase chain reaction. Gene 1998, 210(2):179-186.
• [9]Marco JD, Uezato H, Mimori T, Barroso PA, Korenaga M, Nonaka S, Basombrio MA, Taranto NJ, Hashiguchi Y: Are cytochrome B gene sequencing and polymorphism-specific polymerase chain reaction as reliable as multilocus enzyme electrophoresis for identifying Leishmania spp. from Argentina? Am J Trop Med Hyg 2006, 75(2):256-260.
• [10]Padilla AM, Marco JD, Diosque P, Segura MA, Mora MC, Fernandez MM, Malchiodi EL, Basombrio MA: Canine infection and the possible role of dogs in the transmission of American tegumentary leishmaniosis in Salta, Argentina. Vet Parasitol 2002, 110(1–2):1-10.
• [11]Harris E, Kropp G, Belli A, Rodriguez B, Agabian N: Single-step multiplex PCR assay for characterization of New World Leishmania complexes. J Clin Microbiol 1998, 36(7):1989-1995.
• [12]Evans D: Laboratory maintenance of organisms. In Handbook on Isolation, Characterization, and Cryopreservation of Leishmania. World Health Organization Tech Rep Ser. Edited by Evans D. World Health Organization, Geneva; 1989:12-14.
• [13]: Report of a meeting of the WHO Expert Committee on the Control of Leishmaniases. World Health Organization, Geneva; 2010.
• [14]Eresh S, McCallum SM, Barker DC: Identification and diagnosis of Leishmania mexicana complex isolates by polymerase chain reaction. Parasitology 1994, 109(Pt 4):423-433.
• [15]Shuto T, Furuta T, Oba M, Xu H, Li JD, Cheung J, Gruenert DC, Uehara A, Suico MA, Okiyoneda T, et al.: Promoter hypomethylation of Toll-like receptor-2 gene is associated with increased proinflammatory response toward bacterial peptidoglycan in cystic fibrosis bronchial epithelial cells. FASEB J 2006, 20(6):782-784.
• [16]Santiago Perez MI, Hervada Vidal X, Naveira Barbeito G, Silva LC, Farinas H, Vazquez E, Bacallao J, Mujica OJ: The Epidat program. Rev Panam Salud Publica 2010, 27(1):80-82.
• [17]Azzimonti Renzo JC: Capítulo 4: Índices Clínicos y Estadígrafos. In Bioestadística aplicada a Bioquímica y Farmacia. Second edition edition. Edited by Azzimonti Renzo JC. Editorial Universitaria de la UNaM, Misiones, Argentina; 2003:2-3.
• [18]Bensoussan E, Nasereddin A, Jonas F, Schnur LF, Jaffe CL: Comparison of PCR assays for diagnosis of cutaneous leishmaniasis. J Clin Microbiol 2006, 44(4):1435-1439.
• [19]Rotureau B, Ravel C, Couppie P, Pratlong F, Nacher M, Dedet JP, Carme B: Use of PCR-restriction fragment length polymorphism analysis to identify the main new world Leishmania species and analyze their taxonomic properties and polymorphism by application of the assay to clinical samples. J Clin Microbiol 2006, 44(2):459-467.
• [20]Barrio A, Mora MC, Ramos F, Moreno S, Samson R, Basombrio MA: Use of kDNA-based polymerase chain reaction as a sensitive and differentially diagnostic method of American Tegumentary Leishmaniasis in disease-endemic areas of northern Argentina. Am J Trop Med Hyg 2007, 77(4):636-639.
• [21]Matsumoto T, Hashiguchi Y, Gomez EA, Calvopina MH, Nonaka S, Saya H, Mimori T: Comparison of PCR results using scrape/exudate, syringe-sucked fluid and biopsy samples for diagnosis of cutaneous leishmaniasis in Ecuador. Trans R Soc Trop Med Hyg 1999, 93(6):606-607.
• [22]Mimori T, Matsumoto T, Calvopina MH, Gomez EA, Saya H, Katakura K, Nonaka S, Shamsuzzaman SM, Hashiguchi Y: Usefulness of sampling with cotton swab for PCR-diagnosis of cutaneous leishmaniasis in the New World. Acta Trop 2002, 81(3):197-202.
• [23]Krolewiecki AJ, Romero HD, Cajal SP, Abraham D, Mimori T, Matsumoto T, Juarez M, Taranto NJ: A randomized clinical trial comparing oral azithromycin and meglumine antimoniate for the treatment of American cutaneous leishmaniasis caused by Leishmania (Viannia) braziliensis. Am J Trop Med Hyg 2007, 77(4):640-646.
• [24]Minodier P, Parola P: Cutaneous leishmaniasis treatment. Travel Med Infect Dis 2007, 5(3):150-158.
• [25]Barrio A, Parodi CM, Locatelli F, Mora MC, Basombrio MA, Korenaga M, Hashiguchi Y, Garcia Bustos MF, Gentile A, Marco JD: Leishmania infantum and human visceral leishmaniasis, Argentina. Emerg Infect Dis 2012, 18(2):354-355.