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

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

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
, Kamila Gaudêncio da Silva Sales
, Lucas Lisboa Nunes Bonifácio
, Luciana Aguiar Figueredo
, Thomas R. Shelite
, Fábio dos Santos Nogueira
, Domenico Otranto
, Filipe Dantas-Torres^*

^*Corresponding author for this work

Research output: Journal Publications and Reviews › RGC 21 - Publication in refereed journal › peer-review

1 Citation (Scopus)

9 Downloads (CityUHK Scholars)

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. © The Author(s) 2025.

Original language	English
Article number	2
Journal	Parasites and Vectors
Volume	18
Online published	7 Jan 2025
DOIs	https://doi.org/10.1186/s13071-024-06633-7
Publication status	Published - 2025
Externally published	Yes

Research Keywords

Isothermal amplification
Leishmaniasis
Molecular diagnosis
Point-of-care
Recombinase

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This full text is made available under CC-BY 4.0. https://creativecommons.org/licenses/by/4.0/

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@article{ad6045d0c68c4577a4e4576ed90e5894,

title = "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 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. {\textcopyright} The Author(s) 2025.",

keywords = "Isothermal amplification, Leishmaniasis, Molecular diagnosis, Point-of-care, Recombinase",

author = "\{de Luna\}, \{Rafaela Lira Nogueira\} and Sales, \{Kamila Gaud{\^e}ncio da Silva\} and Bonif{\'a}cio, \{Lucas Lisboa Nunes\} and Figueredo, \{Luciana Aguiar\} and Shelite, \{Thomas R.\} and Nogueira, \{F{\'a}bio dos Santos\} and Domenico Otranto and Filipe Dantas-Torres",

year = "2025",

doi = "10.1186/s13071-024-06633-7",

language = "English",

volume = "18",

journal = "Parasites and Vectors",

issn = "1756-3305",

publisher = "BioMed Central Ltd.",

}

Rapid isothermal molecular tests to discriminate between Leishmania braziliensis and Leishmania infantum infections in dogs. / de Luna, Rafaela Lira Nogueira; Sales, Kamila Gaudêncio da Silva; Bonifácio, Lucas Lisboa Nunes et al.
In: Parasites and Vectors, Vol. 18, 2, 2025.

Research output: Journal Publications and Reviews › RGC 21 - Publication in refereed journal › peer-review

TY - JOUR

T1 - Rapid isothermal molecular tests to discriminate between Leishmania braziliensis and Leishmania infantum infections in dogs

AU - de Luna, Rafaela Lira Nogueira

AU - Sales, Kamila Gaudêncio da Silva

AU - Bonifácio, Lucas Lisboa Nunes

AU - Figueredo, Luciana Aguiar

AU - Shelite, Thomas R.

AU - Nogueira, Fábio dos Santos

AU - Otranto, Domenico

AU - Dantas-Torres, Filipe

PY - 2025

Y1 - 2025

N2 - 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. © The Author(s) 2025.

AB - 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. © The Author(s) 2025.

KW - Isothermal amplification

KW - Leishmaniasis

KW - Molecular diagnosis

KW - Point-of-care

KW - Recombinase

UR - https://www.scopus.com/pages/publications/85214224876

UR - https://www.scopus.com/record/pubmetrics.uri?eid=2-s2.0-85214224876&origin=recordpage

U2 - 10.1186/s13071-024-06633-7

DO - 10.1186/s13071-024-06633-7

M3 - RGC 21 - Publication in refereed journal

C2 - 39773298

SN - 1756-3305

VL - 18

JO - Parasites and Vectors

JF - Parasites and Vectors

M1 - 2

ER -

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

Abstract

Research Keywords

Publisher's Copyright Statement

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