Exploring Leishmania infantum cathepsin as a new molecular marker for phylogenetic relationships and visceral leishmaniasis diagnosis

Abstract

BackgroundLeishmania infantum, the etiological agent of visceral leishmaniasis, is a neglected zoonosis that requires validation and standardization of satisfactory diagnostic methodologies. Thus, the aim of the present study was to evaluate the effectiveness of cathepsin L-like protease as a target for making molecular diagnoses and as a phylogenetic marker enabling to understand the intraspecies variations and evolutionary history of L. infantum in Brazil.MethodsWe used 44 isolates of L. infantum. The cathepsin L-like gene fragments were amplified, sequenced, manually aligned and analyzed using inference methods. The sequences generated were used to search and design oligonucleotide primers to be used in reactions specific to the target parasite.ResultsThe cathepsin L-like gene did not show any intraspecies variability among the isolates analyzed. The pair of primers proposed amplified the target deoxyribonucleic acid (DNA) of L. infantum isolates and were effective for DNA amplification at concentrations of as low as 10− 11 ng/μl. The proposed marker did not present cross-reactions with other hemoparasites. When used for making the diagnosis in a panel of clinical samples from dogs, a positivity rate of 49.03% (102/208) was obtained, versus 14.42% (30/208) for a ribosomal internal transcribed spacer (ITS) marker. In samples from sandflies, the rate was 6.25% and from humans, 14.28%.ConclusionsThe results described in this work allow us to infer that CatLeish-PCR is a sensitive and specific marker for use in diagnostic trials of L. infantum and in clinical and epidemiological surveys.

First molecular-based detection study of Leishmania infantum in the Tapirapé indigenous population in the Brazilian Amazon

“…This study suggests that specific molecular tests for Leishmania infantum may be an alternative to the serological methods commonly used and recommended by health agencies, as they are important tools for solving problems of sensitivity and specificity of serological tests (25). Therefore, this molecular diagnostic test can be efficiently used in routine diagnosis of infections in patients and in epidemiological surveys involving humans, animal hosts, and insect vectors (7).…”

“…The different serological tests used may vary in sensitivity and specificity, depending on the method (26). Importantly, the higher percentage in dogs and human reaction to L. amazonensis may be due to discrepancies in these serological tests, such as specificity failures that induce serum cross-reactivity phenomena (27,28) and the molecular diagnosis based on cathepsin L-like being specific to L. infantum (7).…”

“…For specific molecular diagnosis of Leishmania infantum, PCR was performed using cathepsin L-like gene (7).…”

“…Leishmaniasis is caused by Leishmania infantum (3,4), and the transmission cycle involves a sand fly vector belonging to the Lutzomyia genus of Phlebotominae subfamily (5) and a variety of vertebrate hosts. The domestic dog is the main reservoir, and wild mammals such as marsupials, bats, horses, and felines are hosts of secondary epidemiological importance (6)(7)(8)(9)(10)(11).…”

>b<Diversidade de parasitas dos gêneros >i<Trypanosoma e Leishmania>/i< em vertebrados silvestres e vetores da área da Sede Reserva Legado das Águas- Votorantim, no município de Miracatu, São Paulo>/b<

“…Esta proximidade infere a recente especiação entre as duas, além de demonstrar que a recente introdução da espécie no Continente pode ser embasada pela inexistência de variabilidade genética recentemente demonstrada entre isolados de diversos Biomas do Brasil. (Silva et al, 2019). Separadamente destas, nas regiões asiáticas do Nepal, Paquistão, Bangladesh e Índia, o agente causador da Leishmaniose Visceral é a L. (Leishmania) donovani.…”