Real-Time PCR Assay for detection and quantification of Leishmania: standardization, positive control, validation, and intra-laboratory assay
Manuel Hospinal-Santiani, Carlos Ricardo Soccol, Susan Grace Karp, Eduardo Scopel Ferreira da Costa, Luiz Alberto Junior Letti, Germana Davila dos Santos, Vanete Thomaz Soccol
Abstract
His study aimed to develop a method to investigate PCR sensitivity for diagnosis and ensure reproducibility for parasite load quantification in tissues based on qPCR. In the first step, genes were selected to quantify the parasite load; then, a standard was developed to quantify the concentration of different Leishmania species. These tools were evaluated in intra-laboratory assays. The sensitivity was determined as 0.01 parasites/μL, and the method was reproducible with 100% concordance among human participants in the intra-laboratory validation study. Furthermore, the results demonstrated the specificity of the method in detecting the genus Leishmania without showing cross-reaction with Trypanosoma cruzi or human DNA.
Keywords
References
Akhoundi, M., Downing, T., Votýpka, J., Kuhls, K., Lukeš, J., Cannet, A., Ravel, C., Marty, P., Delaunay, P., Kasbari, M., Granouillac, B., Gradoni, L., & Sereno, D. (2017). Leishmania infections: Molecular targets and diagnosis. Molecular Aspects of Medicine, 57, 1-29. http://dx.doi.org/10.1016/j.mam.2016.11.012. PMid:28159546.
Antinori, S., Calattini, S., Piolini, R., Longhi, E., Bestetti, G., Cascio, A., Parravicini, C., & Corbellino, M. (2009). Is real-time polymerase chain reaction (PCR) more useful than a conventional PCR for the clinical management of leishmaniasis? The American Journal of Tropical Medicine and Hygiene, 81(1), 46-51. http://dx.doi. org/10.4269/ajtmh.2009.81.46. PMid:19556565.
Araujo-Pereira, T., Pita-Pereira, D., Moreira, R. B., Silva-Galdino, T., Duarte, M. P. de O., Brazil, R. P., & Britto, C. (2018). Molecular diagnosis of cutaneous leishmaniasis in an endemic area of Acre State in the Amazonian region of Brazil. Revista da Sociedade Brasileira de Medicina Tropical, 51(3), 376-381. http://dx.doi. org/10.1590/0037-8682-0232-2017. PMid:29972573.
Baneth, G., & Solano-Gallego, L. (2022). Leishmaniasis. The Veterinary Clinics of North America. Small Animal Practice, 52(6), 1359-1375. http://dx.doi.org/10.1016/j.cvsm.2022.06.012. PMid:36336425.
Bensoussan, E., Nasereddin, A., Jonas, F., Jaffe, C. L., & Schnur, L. F. (2006). Comparison of PCR assays for diagnosis of cutaneous leishmaniasis. Journal of Clinical Microbiology, 44(4), 1435- 1439. http://dx.doi.org/10.1128/JCM.44.4.1435-1439.2006. PMid:16597873.
Brito, R. C. F., Aguiar-Soares, R. D. O., Cardoso, M. D. O. J., Coura- Vital, W., Roatt, B. M., & Reis, A. B. (2020). Recent advances and new strategies in leishmaniasis diagnosis. Applied Microbiology and Biotechnology, 104(19), 8105-8116. http://dx.doi.org/10.1007/ s00253-020-10846-y. PMid:32845368.
Castilho, T. M., Shaw, J. J., & Floeter-Winter, L. M. (2003). New PCR assay using glucose-6-phosphate dehydrogenase for identification of Leishmania species. Journal of Clinical Microbiology, 41(2), 540-546. http://dx.doi.org/10.1128/JCM.41.2.540-546.2003. PMid:12574243.
Ceccarelli, M., Galluzzi, L., Diotallevi, A., Andreoni, F., Fowler, H., Petersen, C., Vitale, F., & Magnani, M. (2017). The use of kDNA minicircle subclass relative abundance to differentiate between Leishmania (L.) infantum and Leishmania (L.) amazonensis. Parasites & Vectors, 10(1), 239. http://dx.doi.org/10.1186/ s13071-017-2181-x. PMid:28511704.
Ceccarelli, M., Galluzzi, L., Migliazzo, A., & Magnani, M. (2014). Detection and characterization of Leishmania (Leishmania) and Leishmania (Viannia) by SYBR green-based real-time PCR and high-resolution melt analysis targeting kinetoplast minicircle DNA. PLoS One, 9(2), e88845. http://dx.doi.org/10.1371/journal. pone.0088845. PMid:24551178.
Filgueira, C. P. B., Moreira, O. C., Cantanhêde, L. M., de Farias, H. M. T., Porrozzi, R., Britto, C., Boité, M. C., & Cupolillo, E. (2020). Comparison and clinical validation of qPCR assays targeting Leishmania 18s rDNA and HSP70 genes in patients with American tegumentary leishmaniasis. PLoS Neglected Tropical Diseases, 14(10), e0008750. http://dx.doi.org/10.1371/journal. pntd.0008750. PMid:33044986.
Fraga, J., Montalvo, A. M., de Doncker, S., Dujardin, J. C., & van der Auwera, G. (2010). Phylogeny of Leishmania species based on the heat-shock protein 70 gene. Infection, Genetics and Evolution, 10(2), 238-245. http://dx.doi.org/10.1016/j.meegid.2009.11.007. PMid:19913110.
Froger, A., & Hall, J. E. (2007). Transformation of Plasmid DNA into E. coli using the heat shock method. Journal of Visualized Experiments, 6(6), 253. PMid:18997900.
Fu, J., Li, D., Xia, S., Song, H., Dong, Z., Chen, F., Sun, X., & Tang, Z. (2009). Absolute quantification of plasmid DNA by real-time PCR with genomic DNA as external standard and its application to a biodistribution study of an HIV DNA vaccine. Analytical Sciences: The International Journal of the Japan Society for Analytical Chemistry, 25(5), 675-680. http://dx.doi.org/10.2116/ analsci.25.675. PMid:19430152.
Green, M. R., & Sambrook, J. (2016). Preparation of plasmid DNA by alkaline lysis with sodium dodecyl sulfate: Minipreps. Cold Spring Harbor Protocols, 2016(10), 93344. http://dx.doi.org/10.1101/ pdb.prot093344. PMid:27698243.
Jara, M., Adaui, V., Valencia, B. M., Martinez, D., Alba, M., Castrillon, C., Cruz, M., Cruz, I., Van Der Auwera, G., Llanos-Cuentas, A., Dujardin, J. C., & Arevalo, J. (2013). Real-time PCR assay for detection and quantification of Leishmania (Viannia) organisms in skin and mucosal lesions: Exploratory study of parasite load and clinical parameters. Journal of Clinical Microbiology, 51(6), 1826- 1833. http://dx.doi.org/10.1128/JCM.00208-13. PMid:23554201.
Kumari, D., Perveen, S., Sharma, R., & Singh, K. (2021). Advancement in leishmaniasis diagnosis and therapeutics: An update. European Journal of Pharmacology, 910, 174436. http://dx.doi. org/10.1016/j.ejphar.2021.174436. PMid:34428435.
Kumari D, Mahajan S, Kour P, Singh K. Virulence factors of Leishmania parasite: their paramount importance in unraveling novel vaccine candidates and therapeutic targets. Life Sci. 2022 Oct 1;306:120829. http://dx.doi.org/10.1016/j.lfs.2022.120829. Epub 2022 Jul 22. PMID: 35872004.
Larkin, M. A., Blackshields, G., Brown, N. P., Chenna, R., McGettigan, P. A., McWilliam, H., Valentin, F., Wallace, I. M., Wilm, A., Lopez, R., Thompson, J. D., Gibson, T. J., & Higgins, D. G. (2007). Clustal W and Clustal X version 2.0. Bioinformatics (Oxford, England), 23(21), 2947-2948. http://dx.doi.org/10.1093/bioinformatics/ btm404. PMid:17846036.
Mary, C., Faraut, F., Lascombe, L., & Dumon, H. (2004). Quantification of Leishmania infantum DNA by a real-time pcr assay with high sensitivity. Journal of Clinical Microbiology, 42(11), 5249- 5255. http://dx.doi.org/10.1128/JCM.42.11.5249-5255.2004. PMid:15528722.
Mota, T. F., Brodskyn, C. I., Morello, L. G., Marchini, F. K., Krieger, M. A., Rampazzo, C. P. R., & Fraga, D. B. M. (2022). Multiplex qPCR assay to determine Leishmania infantum load in Lutzomyia longipalpis sandfly samples. Medical and Veterinary Entomology, 36(2), 176-184. http://dx.doi.org/10.1111/mve.12564. PMid:35089617.
Ostolin, T. L. V. P., Gusmão, M. R., Mathias, F. A., Cardoso, J. M. O., Roatt, B. M., Aguiar-Soares, R. O., Ruiz, J. C., Resende, D. M., Brito, R. C. F., & Reis, A. B. (2022). A specific Leishmania infantum polyepitope vaccine triggers Th1-type immune response and protects against experimental visceral leishmaniasis. Cellular Immunology, 380, 104592. http://dx.doi.org/10.1016/j. cellimm.2022.104592. PMid:36084402.
Piron, M., Fisa, R., Casamitjana, N., López-Chejade, P., Puig, L., Vergés, M., Gascón, J., Prat, J. G., Portús, M., & Sauleda, S. (2007). Development of a real-time PCR assay for Trypanosoma cruzi detection in blood samples. Acta Tropica, 103(3), 195- 200. http://dx.doi.org/10.1016/j.actatropica.2007.05.019. PMid:17662227.
Sambrook, J., & Russell, D. W. (2001). Molecular cloning: A laboratory manual. Cold Spring Harbor Laboratory Press.
Schönian, G., Nasereddin, A., Dinse, N., Schweynoch, C., Schallig, H. D. F. H., Presber, W., & Jaffe, C. L. (2003). PCR diagnosis and characterization of Leishmania in local and imported clinical samples. Diagnostic Microbiology and Infectious Disease, 47(1), 349-358. http://dx.doi.org/10.1016/S0732-8893(03)00093-2. PMid:12967749.
Simpson, L., Douglass, S. M., Lake, J. A., Pellegrini, M., & Li, F. (2015). Comparison of the mitochondrial genomes and steady state transcriptomes of two strains of the trypanosomatid parasite, Leishmania tarentolae. PLoS Neglected Tropical Diseases, 9(7), e0003841. http://dx.doi.org/10.1371/journal.pntd.0003841. PMid:26204118.
Tabbabi, A., Cáceres, A. G., Bustamante Chauca, T. P., Seki, C., Choochartpong, Y., Mizushima, D., Yamamoto, D. S., Hashiguchi, Y., & Kato, H. (2020). Nuclear and kinetoplast DNA analyses reveal genetically complex Leishmania strains with hybrid and mito-nuclear discordance in Peru. PLoS Neglected Tropical Diseases, 14(10), e0008797. http://dx.doi.org/10.1371/journal. pntd.0008797. PMid:33075058.
Tandon, R., Reyaz, E., Roshanara, Jadhav, M., Gandhi, M., Dey, R., Salotra, P., Nakhasi, H. L., & Selvapandiyan, A. (2023). Identification of protein biomarkers of attenuation and immunogenicity of centrin or p27 gene deleted live vaccine candidates of Leishmania against visceral leishmaniasis. Parasitology International, 92, 102661. http://dx.doi. org/10.1016/j.parint.2022.102661. PMid:36049661.
Thomaz, C., Mello, C. X., Espíndola, O. M., Shubach, A. O., Quintella, L. P., Oliveira, R. V. C., Duarte, A. C. G., Pimentel, M. I. F., Lyra, M. R., & Marzochi, M. C. (2021). Comparison of parasite load by qPCR and histopathological changes of inner and outer edge of ulcerated cutaneous lesions of cutaneous leishmaniasis. PLoS One, 16(1), e0243978. http://dx.doi.org/10.1371/journal. pone.0243978. PMid:33476320.
Torpiano, P., & Pace, D. (2015). Leishmaniasis: Diagnostic issues in Europe. Expert Review of Anti-Infective Therapy, 13(9), 1123-1138. http://dx.doi.org/10.1586/14787210.2015.105616 0. PMid:26211945.
Weirather, J. L., Jeronimo, S. M. B., Gautam, S., Sundar, S., Kang, M., Kurtz, M. A., Haque, R., Schriefer, A., Talhari, S., Carvalho, E. M., Donelson, J. E., & Wilson, M. E. (2011). Serial quantitative PCR assay for detection, species discrimination, and quantification of Leishmania spp. in human samples. Journal of Clinical Microbiology, 49(11), 3892-3904. http://dx.doi.org/10.1128/ JCM.r00764-11. PMid:22042830.
Wu, Y., Tian, X., Song, N., Huang, M., Wu, Z., Li, S., Waterfield, N. R., Zhan, B., Wang, L., & Yang, G. (2020). Application of quantitative PCR in the diagnosis and evaluating treatment efficacy of leishmaniasis. Frontiers in Cellular and Infection Microbiology, 10, 581639. http://dx.doi.org/10.3389/fcimb.2020.581639. PMid:33117735.
Ye, J., Coulouris, G., Zaretskaya, I., Cutcutache, I., Rozen, S., & Madden, T. L. (2012). Primer-BLAST: A tool to design target-specific primers for polymerase chain reaction. BMC Bioinformatics, 13(1), 134. http://dx.doi.org/10.1186/1471-2105-13-134. PMid:22708584.
Submitted date:
01/31/2023
Accepted date:
06/21/2023
Facebook
Google+
Twitter
LinkedIn
Mendeley
StumbleUpon
CiteULike
Reddit
Email