Rapid isothermal molecular tests to discriminate between Leishmania braziliensis and Leishmania infantum infections in dogs
Abstract Background We standardized two recombinase polymerase amplification (RPA) assays coupled with lateral flow (LF) strips for the detection of Leishmania braziliensis and Leishmania infantum kinetoplast DNA (kDNA). Methods The RPA-LF assays were tested at different temperatures and reaction ti...
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BMC
2025-01-01
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| Series: | Parasites & Vectors |
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| Online Access: | https://doi.org/10.1186/s13071-024-06633-7 |
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| author | Rafaela Lira Nogueira de Luna Kamila Gaudêncio da Silva Sales Lucas Lisboa Nunes Bonifácio Luciana Aguiar Figueredo Thomas R. Shelite Fábio dos Santos Nogueira Domenico Otranto Filipe Dantas-Torres |
| author_facet | Rafaela Lira Nogueira de Luna Kamila Gaudêncio da Silva Sales Lucas Lisboa Nunes Bonifácio Luciana Aguiar Figueredo Thomas R. Shelite Fábio dos Santos Nogueira Domenico Otranto Filipe Dantas-Torres |
| author_sort | Rafaela Lira Nogueira de Luna |
| collection | DOAJ |
| description | Abstract Background We standardized two recombinase polymerase amplification (RPA) assays coupled with lateral flow (LF) strips for the detection of Leishmania braziliensis and Leishmania infantum kinetoplast DNA (kDNA). Methods The RPA-LF assays were tested at different temperatures and reaction times, using DNA from cultured L. braziliensis and L. infantum. The L. infantum RPA-LF was also tested using clinical samples (bone marrow and skin) from infected and uninfected dogs. Results The detection limits (analytical sensitivity) of the assays were 0.04 pg/μl and 0.04 ng/μl for L. braziliensis and L. infantum kDNA, respectively. Using clinical samples, the L. infantum RPA-LF successfully detected the parasite kDNA in bone marrow (21/30; 70.0%) and skin samples (23/30, 76.6%) from naturally infected dogs. We found an almost perfect agreement (kappa = 0.807) between RPA-LF for L. infantum and our reference quantitative real-time polymerase chain reaction (qPCR), considering clinical samples with a quantification cycle (C q) < 30, whereas the agreement with samples with a C q > 30 (lower parasite loads) was moderate (kappa = 0.440). Conclusions The RPA-LF assays developed here may be promising diagnostic tools for point-of-care diagnosis of L. infantum and L. braziliensis infection in dogs, particularly in remote rural areas lacking laboratory infrastructure. Graphical Abstract |
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| id | doaj-art-c2daf4b43431434d9cc67e8a62f2c865 |
| institution | Kabale University |
| issn | 1756-3305 |
| language | English |
| publishDate | 2025-01-01 |
| publisher | BMC |
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| series | Parasites & Vectors |
| spelling | doaj-art-c2daf4b43431434d9cc67e8a62f2c8652025-01-12T12:09:57ZengBMCParasites & Vectors1756-33052025-01-011811910.1186/s13071-024-06633-7Rapid isothermal molecular tests to discriminate between Leishmania braziliensis and Leishmania infantum infections in dogsRafaela Lira Nogueira de Luna0Kamila Gaudêncio da Silva Sales1Lucas Lisboa Nunes Bonifácio2Luciana Aguiar Figueredo3Thomas R. Shelite4Fábio dos Santos Nogueira5Domenico Otranto6Filipe Dantas-Torres7Aggeu Magalhães Institute, Oswaldo Cruz Foundation (Fiocruz)Aggeu Magalhães Institute, Oswaldo Cruz Foundation (Fiocruz)Aggeu Magalhães Institute, Oswaldo Cruz Foundation (Fiocruz)Aggeu Magalhães Institute, Oswaldo Cruz Foundation (Fiocruz)Department of Biosafety, University of Texas Medical BranchAndradina Educational FoundationDepartment of Veterinary Medicine, University of BariAggeu Magalhães Institute, Oswaldo Cruz Foundation (Fiocruz)Abstract Background We standardized two recombinase polymerase amplification (RPA) assays coupled with lateral flow (LF) strips for the detection of Leishmania braziliensis and Leishmania infantum kinetoplast DNA (kDNA). Methods The RPA-LF assays were tested at different temperatures and reaction times, using DNA from cultured L. braziliensis and L. infantum. The L. infantum RPA-LF was also tested using clinical samples (bone marrow and skin) from infected and uninfected dogs. Results The detection limits (analytical sensitivity) of the assays were 0.04 pg/μl and 0.04 ng/μl for L. braziliensis and L. infantum kDNA, respectively. Using clinical samples, the L. infantum RPA-LF successfully detected the parasite kDNA in bone marrow (21/30; 70.0%) and skin samples (23/30, 76.6%) from naturally infected dogs. We found an almost perfect agreement (kappa = 0.807) between RPA-LF for L. infantum and our reference quantitative real-time polymerase chain reaction (qPCR), considering clinical samples with a quantification cycle (C q) < 30, whereas the agreement with samples with a C q > 30 (lower parasite loads) was moderate (kappa = 0.440). Conclusions The RPA-LF assays developed here may be promising diagnostic tools for point-of-care diagnosis of L. infantum and L. braziliensis infection in dogs, particularly in remote rural areas lacking laboratory infrastructure. Graphical Abstracthttps://doi.org/10.1186/s13071-024-06633-7Isothermal amplificationLeishmaniasisMolecular diagnosisPoint-of-careRecombinase |
| spellingShingle | Rafaela Lira Nogueira de Luna Kamila Gaudêncio da Silva Sales Lucas Lisboa Nunes Bonifácio Luciana Aguiar Figueredo Thomas R. Shelite Fábio dos Santos Nogueira Domenico Otranto Filipe Dantas-Torres Rapid isothermal molecular tests to discriminate between Leishmania braziliensis and Leishmania infantum infections in dogs Parasites & Vectors Isothermal amplification Leishmaniasis Molecular diagnosis Point-of-care Recombinase |
| title | Rapid isothermal molecular tests to discriminate between Leishmania braziliensis and Leishmania infantum infections in dogs |
| title_full | Rapid isothermal molecular tests to discriminate between Leishmania braziliensis and Leishmania infantum infections in dogs |
| title_fullStr | Rapid isothermal molecular tests to discriminate between Leishmania braziliensis and Leishmania infantum infections in dogs |
| title_full_unstemmed | Rapid isothermal molecular tests to discriminate between Leishmania braziliensis and Leishmania infantum infections in dogs |
| title_short | Rapid isothermal molecular tests to discriminate between Leishmania braziliensis and Leishmania infantum infections in dogs |
| title_sort | rapid isothermal molecular tests to discriminate between leishmania braziliensis and leishmania infantum infections in dogs |
| topic | Isothermal amplification Leishmaniasis Molecular diagnosis Point-of-care Recombinase |
| url | https://doi.org/10.1186/s13071-024-06633-7 |
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