Comparación de pruebas moleculares para el diagnóstico de Leishmania spp. en lesiones cutáneas con baja carga parasitaria
[Comparison of molecular tests for the diagnosis of Leishmania spp. in skin lesions with low parasite load]Adelys Reina1, Vanessa Pineda1, José Eduardo Calzada1, Azael Saldaña Patiño2
1. Departamento de Investigaciones en Parasitología, Instituto Conmemorativo Gorgas de Estudios de la Salud, Panamá, Rep. de Panamá; 2. Centro de Investigación y Diagnóstico de Enfermedades Parasitarias, Facultad de Medicina, Universidad de Panamá, Panamá, Rep. de Panamá.
Descargas
Archivos adicionales
Resumen
Introducción: La leishmaniasis cutánea (LC) es un problema grave de salud pública en Panamá. El diagnóstico de esta parasitosis ha sido siempre desafiante, no sólo debido a su similitud con otras infecciones dérmicas, sino también a características particulares de las lesiones, como cargas parasitarias bajas. Materiales y Métodos: En este estudio, se evaluaron mediante cuatro métodos moleculares, 235 muestras de ADN procedentes de lesiones con frotis negativos por LC obtenidas durante el período 2015-2019. Resultados: Los resultados señalan que las sensibilidades encontradas fueron de 75.6% (IC 0.6234-0.8709) para la PCR kDNA-Género específico, de 66.7% (IC 0.5359-0.776) para la PCR Hsp70-Género específico y de 77.6% (IC 0.645-0.8949) para la qPCR 18S ribosomal. Todas las pruebas obtuvieron un valor predictivo positivo de 100%, mientras que el valor predictivo negativo más alto fue con la qPCR (80.58%) y el más bajo con el PCR Hsp70-Género específico (73.2%). En cuanto a la precisión de diagnóstico se obtuvo un rango mayor del 82% en todas las pruebas evaluadas. Conclusión: Este estudio confirma la buena sensibilidad de la PCR kDNA-Viannia para el análisis de lesiones de LC con baja carga parasitaria. Esta metodología es relativamente fácil de estandarizar, por lo que se recomienda su uso en laboratorios clínicos regionales de Panamá. Aun cuando la qPCR 18S ribosomal presentó una sensibilidad relativamente menor, el uso de esta metodología debe ser también considerada por su facilidad de uso, menor tiempo de ejecución y capacidad de cuantificación.
Abstract
Introduction: Cutaneous leishmaniasis (CL) is a serious public health problem in Panama. The diagnosis of this parasitosis has always been challenging, not only because of its similarity to other dermal infections, but also because of characteristics of the lesions, such as low parasite loads. Materials and Methods: In this study, 235 DNA samples from smear-negative lesions by LC obtained during the period 2015-2019 were evaluated by four molecular methods. Results: The results indicate that the sensitivities found were 75.6% (CI 0.6234-0.8709) for kDNA-Gene-specific PCR, 66.7% (CI 0.5359-0.776) for Hsp70-Gene-specific PCR and 77.6% (CI 0.645-0.8949) for 18S ribosomal qPCR. All tests obtained a positive predictive value of 100%, while the highest negative predictive value was with qPCR (80.58%) and the lowest with Hsp70-Gene-specific PCR (73.2%). In terms of diagnostic accuracy, a range greater than 82% was obtained in all the tests evaluated. Conclusion: This study confirms the good sensitivity of kDNA-Viannia PCR for the analysis of LC lesions with low parasite load. This methodology is relatively easy to standardize, so it is recommended for use in regional clinical laboratories in Panama. Although 18S ribosomal qPCR showed a relatively lower sensitivity, the use of this methodology should also be considered because of its ease of use, shorter execution time and quantification capacity.
Citas
[1] Steverding D. The history of leishmaniasis. Parasites and Vectors. 2017;10(1):1-10. URL: https://doi.org/10.1186/s13071-017-2028-5
[2] Roatt BM, de Oliveira Cardoso JM, De Brito RCF, Coura-Vital W, de Oliveira Aguiar-Soares RD, Reis AB. Recent advances and new strategies on leishmaniasis treatment. Appl Microbiol Biotechnol. 2020;104(21):8965-77.URL: https://doi.org/10.1007/s00253-020-10856-w
[3] Mann S, Frasca K, Scherrer S, Henao-Martínez AF, Newman S, Ramanan P, et al. A Review of Leishmaniasis: Current Knowledge and Future Directions. Curr Trop Med Reports. 2021;8(2):121-32. URL: https://doi.org/10.1007/s40475-021-00232-7
[4] Çizmeci Z, Karakuş M, Karabela ŞN, Erdoğan B, Güleç N. Leishmaniasis in Istanbul; A new epidemiological data about refugee leishmaniasis. Acta Trop. 2019;195(April):23-7. URL:https://doi.org/10.1016/j.actatropica.2019.04.008
[5] OPS. Manual de procedimientos para la vigilancia y control de las leishmaniasis en las Américas [Internet]. Washington, D.C.; 2019. 166 p. Available from: www.paho.org
[6] Davila M, Pineda V, Calzada JE, Saldaña A, Samudio F. Evaluation of cytochrome b sequence to identify leishmania species and variants: The case of Panama. Mem Inst Oswaldo Cruz. 2021;116(1).URL: https://doi.org/10.1590/0074-02760200572
[7] Rigg CA, Calzada JE, Saldaña A, Perea M, Chaves LF, Valderrama A. Leishmania spp. infection rate and feeding patterns of sand flies (diptera: Psychodidae) from a hyperendemic cutaneous leishmaniasis community in Panamá. Am J Trop Med Hyg [Internet]. 2019 Apr [cited 2019 Oct 3];100(4):798-807. Available from: http://www.ncbi.nlm.nih.gov/pubmed/30793681URL: https://doi.org/10.4269/ajtmh.17-0628
[8] OPS, OMS, MINSA, ICGES C. Guía para el abordaje integral de la leishmaniasis en Panamá,2015. 2015;1-70.
[9] Moreira OC, Yadon ZE, Cupolillo E. The applicability of real-time PCR in the diagnostic of cutaneous leishmaniasis and parasite quantification for clinical management: Current status and perspectives. Acta Trop [Internet]. 2018;184(August):29-37. Available from: http://dx.doi.org/10.1016/j.actatropica.2017.09.020URL: https://doi.org/10.1016/j.actatropica.2017.09.020
[10] Jaramillo O, Espinoza Aguirre A, Calvo Fonseca N, Mata Somarriba C, Wasserman H. La leishmaniosis cutánea en Costa Rica : prevención , diagnóstico y tratamiento. 2018;103-14.
[11] Filgueira CPB, Moreira OC, Cantanhêde LM, de Farias HMT, Porrozzi R, Britto C, et al. Comparison and clinical validation of QPCR assays targeting leishmania 18s rDNA and HSP70 genes in patients with american tegumentary leishmaniasis. PLoS Negl Trop Dis [Internet]. 2020 Oct 1 [cited 2023 Apr 25];14 (10):1-18. Available from: https://journals.plos.org/plosntds/article?id=10.1371/journal.pntd.0008750. URL: https://doi.org/10.1371/journal.pntd.0008750
[12] Merino G, Rodríguez J, Morillas-Márquez F, Tercedor J, Corpas-López V, Chiheb S, et al. Comparison of PCR-based methods for the diagnosis of cutaneous leishmaniasis in two different epidemiological scenarios: Spain and Morocco. J Eur Acad Dermatology Venereol. 2018;32(11):1999-2003. URL: https://doi.org/10.1111/jdv.15034
[13] Thakur S, Joshi J, Kaur S. Leishmaniasis diagnosis: an update on the use of parasitological, immunological and molecular methods. J Parasit Dis Off Organ Indian Soc Parasitol [Internet]. 2020 Jun 1 [cited 2023 Feb 1];44(2):253. Available from: /pmc/articles/PMC7223249/. URL: https://doi.org/10.1007/s12639-020-01212-w
[14] Ceccarelli M, Buffi G, Diotallevi A, Andreoni F, Bencardino D, Vitale F, et al. Evaluation of a kDNA-Based qPCR Assay for the Detection and Quantification of Old World Leishmania Species. Microorganisms [Internet]. 2020 Dec 16 [cited 2022 Mar 8];8(12):2006. Available from: https://www.mdpi.com/2076-2607/8/12/2006. URL: https://doi.org/10.3390/microorganisms8122006
[15] Weigle KA, Labrada LA, Lozano C, Santrich C, Barker DC. PCR-based diagnosis of acute and chronic cutaneous leishmaniasis caused by Leishmania (Viannia). J Clin Microbiol. 2002;40(2):601-6. URL: https://doi.org/10.1128/JCM.40.2.601-606.2002
[16] Tsokana CN, Athanasiou L V., Valiakos G, Spyrou V, Manolakou K, Billinis C. Molecular Diagnosis of Leishmaniasis, Species Identification and Phylogenetic Analysis. Intech open. 2018;2:64.
[17] Vergel C, Walker J, Saravia NG. Amplification of human DNA by primers targeted to Leishmania kinetoplast DNA and post-genome considerations in the detection of parasites by a polymerase chain reaction. Am J Trop Med Hyg. 2005;72(4):423-9. URL: https://doi.org/10.4269/ajtmh.2005.72.423
[18] Sevilha-Santos L, dos Santos Júnior ACM, Medeiros-Silva V, Bergmann JO, da Silva EF, Segato LF, et al. Accuracy of qPCR for quantifying Leishmania kDNA in different skin layers of patients with American tegumentary leishmaniasis. Clin Microbiol Infect. 2019 Feb 1;25(2):242-7. URL: https://doi.org/10.1016/j.cmi.2018.04.025
[19] Passos VMA, Fernandes O, Lacerda PAF, Volpini AC, Gontijo CMF, Degrave W, et al. Leishmania (Viannia) braziliensis is the predominant species infecting patients with American cutaneous leishmaniasis in the State of Minas Gerais, Southeast Brazil. Acta Trop. 1999;72(3):251-8. URL: https://doi.org/10.1016/S0001-706X(98)00100-4
[20] Volpini ÂC, Passos VMA, Oliveira GC, Romanha AJ. PCR-RFLP to identify Leishmania (Viannia) braziliensis and L. (Leishmania) amazonensis causing American cutaneous leishmaniasis. Acta Trop. 2004; URL: https://doi.org/10.1016/j.actatropica.2003.10.008
[21] Montalvo AM, Fraga J, Monzote L, Montano I, De Doncker S, Dujardin JC, et al. Heat-shock protein 70 PCR-RFLP: A universal simple tool for Leishmania species discrimination in the New and Old World. Parasitology. 2010;137(8):1159-68.URL: https://doi.org/10.1017/S0031182010000089
[22] Montalvo AM, Fraga J, Maes I, Dujardin J-C, Van der Auwera G. Three new sensitive and specific heat-shock protein 70 PCRs for global Leishmania species identification. Eur J Clin Microbiol Infect Dis [Internet]. 2012;31(7):1453-61. Available from: http://link.springer.com/10.1007/s10096-011-1463-z. URL: https://doi.org/10.1007/s10096-011-1463-z
[23] Garcia L, Kindt A, Bermudez H, Llanos-Cuentas A, De Doncker S, Arevalo J, et al. Culture-independent species typing of neotropical Leishmania for clinical validation of a PCR-based assay targeting heat shock protein 70 genes. J Clin Microbiol [Internet]. 2004 May [cited 2022 Aug 26];42(5):2294-7. Available from: https://pubmed.ncbi.nlm.nih.gov/15131217/ URL: https://doi.org/10.1128/JCM.42.5.2294-2297.2004
[24] Van Der Meide W, Guerra J, Schoone G, Farenhorst M, Coelho L, Faber W, et al. Comparison between quantitative nucleic acid sequence-based amplification, real-time reverse transcriptase PCR, and real-time PCR for quantification of Leishmania parasites. J Clin Microbiol. 2008 Jan;46(1):73-8. URL: https://doi.org/10.1128/JCM.01416-07
[25] Armijos T, Sebastián V. Estudio comparativo de métodos moleculares para el diagnóstico de Leishmaniasis Cutánea. 2021 [cited 2023 May 11]; Available from: http://www.dspace.uce.edu.ec/handle/25000/24823
[26] Faber WR, Oskam L, van Gool T, Kroon NCM, Knegt-Junk KJ, Hofwegen H, et al. Value of diagnostic techniques for cutaneous leishmaniasis. J Am Acad Dermatol. 2003 Jul 1;49(1):70-4. URL: https://doi.org/10.1067/mjd.2003.492
[27] Çulha G, Kaya T, Gülbol Duran G, Urhan Küçük M, Doğramaci AÇ, Tiyekli Çelik D. Investigation of polymerase chain reaction method in patients with suspected chronic cutaneous leishmania of negative microscopy. Mikrobiyol Bul. 2019;53(4):408-18. URL: https://doi.org/10.5578/mb.68692
[28] Espir TT, Guerreiro TS, Naiff M de F, Figueira L de P, Soares FV, da Silva SS, et al. Evaluation of different diagnostic methods of American Cutaneous Leishmaniasis in the Brazilian Amazon. Exp Parasitol. 2016 Aug 1;167:1-6. URL: https://doi.org/10.1016/j.exppara.2016.04.010
[29] Thomaz C, Mello CX de, Espíndola O de M, Shubach A de O, Quintella LP, Oliveira RVC de, et al. Comparison of parasite load by qPCR and histopathological changes of inner and outer edge of ulcerated cutaneous lesions of cutaneous leishmaniasis. PLoS One. 2021 Jan 1;16(1 January). URL: https://doi.org/10.1371/journal.pone.0243978
[30] de Paiva-Cavalcanti M, de Morais RCS, Pessoa-e-Silva R, Trajano-Silva LAM, Gonçalves-de-Albuquerque S da C, Tavares D de HC, et al. Leishmaniases diagnosis: An update on the use of immunological and molecular tools [Internet]. Vol. 5, Cell and Bioscience. BioMed Central Ltd.; 2015 [cited 2021 Jun 21]. p. 1-10. Available from: https://link.springer.com/articles/10.1186/s13578-015-0021-2 URL: https://doi.org/10.1186/s13578-015-0021-2
[31] Bensoussan E, Nasereddin A, Jonas F, Schnur LF, Jaffe CL. Comparison of PCR assays for diagnosis of cutaneous leishmaniasis. J Clin Microbiol. 2006 Apr;44(4):1435-9. URL: https://doi.org/10.1128/JCM.44.4.1435-1439.2006
[32] Miranda A, Paz H, Pascale JM, Samudio FE. Molecular Epidemiology of American Tegumentary Leishmaniasis in Panama Characterization of histopathological changes and cell immune response in skin injuries of skin leishmaniosis patients in Panama View project Sandfly from Panamá View project. 2009 [cited 2023 May 11]; Available from: https://www.researchgate.net/publication/26881839
[33] Inga R, De Doncker S, Gomez J, Lopez M, Garcia R, Le Ray D, et al. Relation between variation in copy number of ribosomal RNA encoding genes and size of harbouring chromosomes in Leishmania of subgenus Viannia. Mol Biochem Parasitol. 1998 May 1;92(2):219-28. URL: https://doi.org/10.1016/S0166-6851(98)00009-7
[34] Reina AM, Mewa JC, Calzada JE, Saldaña A. Characterization of Leishmania spp. Causing Cutaneous Lesions with a Negative Parasitological Diagnosis in Panama. Trop Med Infect Dis. 2022;7(10). URL: https://doi.org/10.3390/tropicalmed7100282
[35] Sandoval-Juárez A, Minaya-Gómez G, Rojas-Palomino N, Cáceres O. Identificación de especies de Leishmania en pacientes derivados al Instituto Nacional de Salud del Perú. Rev Peru Med Exp Salud Publica. 2020 Jun 8;37(1):87-92. URL: https://doi.org/10.17843/rpmesp.2020.371.4514
[36] Merdekios B, Pareyn M, Tadesse D, Eligo N, Kassa M, Jacobs BKM, et al. Evaluation of conventional and four real-time pcr methods for the detection of leishmania on field-collected samples in Ethiopia. PLoS Negl Trop Dis [Internet]. 2021 Jan 1 [cited 2022 Mar 6];15(1):1-18. Available from: /pmc/articles/PMC7802924/. URL: https://doi.org/10.1371/journal.pntd.0008903
[37] Jara M, Valencia BM, Adaui V, Alba M, Lau R, Arevalo J, et al. Quantitative Kinetoplast DNA Assessment during Treatment of Mucosal Leishmaniasis as a Potential Biomarker of Outcome: A Pilot Study. Am J Trop Med Hyg. 2016;94(1):107-13. URL: https://doi.org/10.4269/ajtmh.15-0514
[38] Ghosh P, Hasnain MG, Hossain F, Ashfaq Khan MA, Chowdhury R, Faisal K, et al. Evaluation of Real-time PCR for Diagnosis of Post-Kala-azar Dermal Leishmaniasis in Endemic Foci of Bangladesh. Open Forum Infect Dis [Internet]. 2018 Oct 1 [cited 2023 May 11];5(10). Available from: https://academic.oup.com/ofid/article/5/10/ofy234/5098413 URL: https://doi.org/10.1093/ofid/ofy234
[39] Perez J. Carga Parasitaria, Distribución Espacial y Diversidad Genética de Lesihmania spp en Personalde Militar del Ejercito Nacional de Colombia con Lesiones Cutáneas. 2021 [cited 2023 Mar 15];80. Available from: https://repositorio.unbosque.edu.co/handle/20.500.12495/7056
[40] Ibarra-Meneses AV, Chicharro C, Sánchez C, García E, Ortega S, Ndung'u JM, et al. Loop-Mediated Isothermal Amplification Allows Rapid, Simple and Accurate Molecular Diagnosis of Human Cutaneous and Visceral Leishmaniasis Caused by Leishmania infantum When Compared to PCR. Microorganisms [Internet]. 2021; Available from: https://doi.org/10.3390/microorganisms9030610
Licencia
Derechos de autor 2023 Infomedic Intl.Derechos autoriales y de reproducibilidad. La Revista Médica de Panama es un ente académico, sin fines de lucro, que forma parte de la Academia Panameña de Medicina y Cirugía. Sus publicaciones son de tipo acceso gratuito de su contenido para uso individual y académico, sin restricción. Los derechos autoriales de cada artículo son retenidos por sus autores. Al Publicar en la Revista, el autor otorga Licencia permanente, exclusiva, e irrevocable a la Sociedad para la edición del manuscrito, y otorga a la empresa editorial, Infomedic International Licencia de uso de distribución, indexación y comercial exclusiva, permanente e irrevocable de su contenido y para la generación de productos y servicios derivados del mismo. En caso que el autor obtenga la licencia CC BY, el artículo y sus derivados son de libre acceso y distribución.
