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
. 2022 Dec 16:55:e0069-2022.
doi: 10.1590/0037-8682-0069-2022. eCollection 2022.

Sensitivity comparison for the Leishmania spp. detection in different canine tissues using PCR-HRM

Ana Fidelina Gómez Garay  1 , Stefania Fraenkel  1 , Jorge Javier Alfonso Ruiz Diaz  1 , Oscar Daniel Salvioni Recalde  1 , María Celeste Vega Gómez  1 , Jorge Arístides Miret Riquelme  2   3 , Paola Verónica Arze  1 , Gloria Natalia Ramírez Centurión  1 , Milena Britos  1 , Miriam Rolón  1
Affiliations

Sensitivity comparison for the Leishmania spp. detection in different canine tissues using PCR-HRM

Ana Fidelina Gómez Garay et al. Rev Soc Bras Med Trop. .

Abstract

Background: Leishmaniasis is a vector-borne disease caused by a parasite protozoon from the genus Leishmania. Among the molecular techniques applied for detecting these parasites, real-time PCR with High Resolution Melting (PCR-HRM) proved advantageous since it simultaneously determines both the presence and species of the pathogen in one step, through amplification and later analysis of curves generated by melting temperature.

Methods: Based on this molecular technique, the goal of this study was to estimate the PCR-HRM sensitivity for Leishmania spp. detection in different canine tissues by evaluating biological samples obtained from popliteal, submandibular, and pre-scapular lymph nodes, from bone marrow and ear pinnae of 28 stray dogs captured in the metropolitan area of Asunción (Paraguay).

Results: The rk39 immunochromatographic test showed that 25/28 tested dogs (89%) presented antibodies against L. infantum. In 20/25 dogs that tested positive for rk39 (80%), it was possible to detect Leishmania spp. by PCR-HRM and determine that the species corresponded entirely to L. infantum. Regarding the analysis of different tissues, the parasite was detected in all popliteal lymph node samples, followed by high detection in submandibular (at 95%) and pre-scapular lymph nodes (at 90%), bone marrow (at 85%), and ear pinnae (at 85%).

Conclusions: This study demonstrated that the use of real-time PCR-HRM using the molecular marker hsp70 was a highly sensitive method for simultaneously detecting and identifying Leishmania species in different tissues taken from infected dogs. In addition, the usefulness of ear pinnae as easily accessible tissue for molecular diagnosis was emphasized.

PubMed Disclaimer

Conflict of interest statement

Conflict of interest: The authors do not declare any conflict of interests.

Figures

FIGURE 1:
FIGURE 1:. Canis familiaris specimen with dermal injuries and ulcers typical of leishmaniasis.
FIGURE 2:
FIGURE 2:. (A) Tm values obtained with the high-resolution melt (HRM) assay for positive control (L. (L.) infantum) and canine tissue samples. (B) Normalized curve of the heat shock protein (hsp70) amplicon obtained by HRM.
FIGURE 3:
FIGURE 3:. Comparative equivalent parasite DNA in tissues using ANOVA.
SUPPLEMENTARY FIGURE 1:
SUPPLEMENTARY FIGURE 1:. Standard curve target hsp70: threshold cycle values and equivalent parasite DNA by reaction.
See this image and copyright information in PMC

Similar articles

See all similar articles

Cited by

References

    1. World Health Organization . Control of Neglected Tropical Disease. WHO: Geneva Switzerland; 2020. [2021 June 1]. Available from: https://www.who.int/teams/control-of-neglected-tropical-diseases/neglect... .
    1. World Health Organization . Health Topics, Leishmaniasis. WHO: Geneva Switzerland; 2020. [2021 June 1]. Available from: https://www.who.int/health-topics/leishmaniasis#tab=tab_1 .
    1. Handman E, Bullen VR. Interaction of Leishmania with the host macrophage. Trends Parasitol. 2002;18(1):332–334. - PubMed
    1. Panamerican Health Organization . Informe Epidemiológico das Américas. PAHO; Washington USA: 2020. [2021 May 23]. Available from: https://iris.paho.org/handle/10665.2/53091 .
    1. World Health Organization . Leishmaniasis. Key Fact. WHO: Geneva Switzerland; 2020. [2021 June 1]. Available from: https://www.who.int/news-room/fact-sheets/detail/leishmaniasis .

MeSH terms