Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2025 Jan 7;18(1):2.
doi: 10.1186/s13071-024-06633-7.

Rapid isothermal molecular tests to discriminate between Leishmania braziliensis and Leishmania infantum infections in dogs

Rafaela Lira Nogueira de Luna  1 , Kamila Gaudêncio da Silva Sales  1 , Lucas Lisboa Nunes Bonifácio  1 , Luciana Aguiar Figueredo  1 , Thomas R Shelite  2 , Fábio Dos Santos Nogueira  3 , Domenico Otranto  4   5 , Filipe Dantas-Torres  6
Affiliations

Rapid isothermal molecular tests to discriminate between Leishmania braziliensis and Leishmania infantum infections in dogs

Rafaela Lira Nogueira de Luna et al. Parasit Vectors. .

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 (Cq) < 30, whereas the agreement with samples with a Cq > 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.

Keywords: Isothermal amplification; Leishmaniasis; Molecular diagnosis; Point-of-care; Recombinase.

PubMed Disclaimer

Conflict of interest statement

Declarations. Ethics approval and consent to participate: This study used samples obtained from dogs during a previous study, whose procedures were approved by the Ethics Committee on Animal Use of Andradina Educational Foundation (CEUA/FEA nº 0010). The procedures of the current study were also assessed and authorized by the Ethics Committee on Animal Use of Aggeu Magalhães Institute (CEUA nº 153/19). Consent for publication: Not applicable. Competing interests: Filipe Dantas-Torres is the Editor-in-Chief of Parasites & Vectors.

Figures

Fig. 1
Fig. 1
Schematic illustrations of the PCRD FLEX lateral flow (LF) strips (Abingdon Health, York, UK) (A): a, positive result for test line 1 (T1); b, positive result for test line (T2); c, negative result showing control line (C); d, an illustration of all lines (T1 detects DIG/biotin-labeled amplicons; T2 detects FAM/biotin- or FICT/biotin-labeled amplicons; C is the control line). Actual examples of a T1-positive (left) and a T1-negative (right) result with the recombinase polymerase amplification-LF assay (RPA-LF) for L. infantum (B)
Fig. 2
Fig. 2
Representative examples of the recombinase polymerase amplification–lateral flow assays (RPA-LF) for L. braziliensis (A) and L. infantum (B). Standard DNA was obtained from cultured promastigotes of reference strains (see Methods for details), and a master mix without DNA template was used as no-template control (NTC). The assays for L. braziliensis and L. infantum were performed at 45 °C and 40 °C, respectively, for 40 min
Fig. 3
Fig. 3
Results of the recombinase polymerase amplification lateral flow assay (RPA-LF) for L. infantum on DNA extracted from skin samples from naturally infected dogs (numbers 1–5)
See this image and copyright information in PMC

References

    1. Dantas-Torres F, Solano-Gallego L, Baneth G, Ribeiro VM, de Paiva-Cavalcanti M, Otranto D. Canine leishmaniosis in the Old and New Worlds: unveiled similarities and differences. Trends Parasitol. 2012;28:531–8. - DOI - PubMed
    1. Travi BL, Cordeiro-da-Silva A, Dantas-Torres F, Miró G. Canine visceral leishmaniasis: Diagnosis and management of the reservoir living among us. PLoS Negl Trop Dis. 2018;12:e0006082. - DOI - PMC - PubMed
    1. Dantas-Torres F. Canine leishmaniasis in the Americas: etiology, distribution, and clinical and zoonotic importance. Parasit Vectors. 2024;17:198. - DOI - PMC - PubMed
    1. Dantas-Torres F, de Paiva-Cavalcanti M, Figueredo LA, Melo MF, da Silva FJ, da Silva AL, et al. Cutaneous and visceral leishmaniasis in dogs from a rural community in northeastern Brazil. Vet Parasitol. 2010;170:313–7. - DOI - PubMed
    1. Silva DA, Madeira MF, Teixeira AC, de Souza CM, Figueiredo FB. Laboratory tests performed on Leishmania seroreactive dogs euthanized by the leishmaniasis control program. Vet Parasitol. 2011;179:257–61. - DOI - PubMed

MeSH terms

LinkOut - more resources